Tissue selective transgene expression

ABSTRACT

Provided herein are compositions and methods for selective expression of a transgene. Compositions and methods for selective expression of a transgene comprise one or more human regulatory elements, which, when operably linked to a transgene, can facilitate selective expression of a transgene (e.g., cell-type selective expression) in a target cell as compared to at least one or more non-target cells.

CROSS-REFERENCE

This application is a continuation application of InternationalApplication No. PCT/US2018/025940, filed Apr. 3, 2018, which claims thebenefit of U.S. Provisional Application No. 62/480,998, filed Apr. 3,2017, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 29, 2018, isnamed 46482-704_601_SL.txt and is 78,248 bytes in size.

BACKGROUND OF THE DISCLOSURE

Gene therapy has long been recognized for its enormous potential in howwe approach and treat human diseases. Instead of relying on drugs orsurgery, patients, especially those with underlying genetic factors, canbe treated by directly targeting the underlying cause. Furthermore, bytargeting the underlying genetic cause, gene therapy has the potentialto effectively cure patients or provide sustained treatment over alonger period of time. Yet, despite this, clinical applications of genetherapy still require improvement in several aspects. One area ofconcern is off target effects. An attractive approach to address offtarget effects is to target gene expression of gene therapy to celltype(s) or tissue(s) of interest, or the target cell type(s) ortissue(s). As such, there is a need to identify elements and methods ofuse thereof for targeting gene therapy or gene expression to a tissue orcell type of interest.

SUMMARY OF THE DISCLOSURE

There exists a considerable need for targeting gene therapy andgene/transgene expression thereof to the desired tissue and/or cell typein vivo, which can decrease off-target effects, increase therapeuticefficacy in the target tissue and/or cell type, and increase patientsafety and tolerance by lowering the effective dose needed to achieveefficacy.

Provided herein are compositions and methods for selective expression ofa transgene in a target tissue or cell type over one or more non-targettissue or cell types. Compositions and methods for selective expressionof a transgene comprise one or more regulatory elements (REs) which,when operably linked to a transgene (e.g., an ion channel subunit or aneurotransmitter regulator, or a syntaxin-binding protein), canfacilitate or result in selective or preferential expression of thetransgene in a target tissue or cell type (e.g., parvalbumin (PV)neurons) as compared to one or more non-target cell types (e.g., non-PVcells). In some cases, the REs are non-naturally occurring sequences. Insome cases, the REs are human-derived regulatory elements. In somecases, the REs comprise a sequence from a non-human species, such as amonkey, or a dog, or a rabbit, or a mouse. In some cases, thecompositions described herein are delivered into a cell in vivo, exvivo, or in vitro using a viral vector and/or virus particles, such asadeno-associated virus (AAV) or lentivirus. In some cases, thecompositions described herein are delivered into a cell as gene therapy.Also contemplated herein are methods and compositions for treating aneurological condition or disorder associated with a genetic defect inthe CNS. In some cases, the relevant cell type or tissue affected by thegenetic defect is a PV cell. In some instances, the neurologicalcondition or disease is Dravet syndrome, Alzheimer's disease, epilepsy,and/or seizures. In some cases, the neurological condition or disease isa psychiatric disorder (e.g., schizophrenia, obsessive compulsivedisorder, addiction, depression, anxiety, psychosis); an autism spectrumdisorder (e.g., Fragile X syndrome, Rett syndrome); epilepsy (e.g.,chronic traumatic encephalopathy, generalized epilepsy with febrileseizures plus (GEFS+), epileptic encephalopathy, temporal lobe epilepsy,focal epilepsy, tuberous sclerosis); or neurodegeneration (e.g.,Alzheimer's disease, Parkinson's disease). In some cases, theneurological condition or disease is any seizure and/or epilepsy relatedcondition or disease wherein PV neurons are implicated.

In one aspect, the present disclosure contemplates a nucleic acidcassette comprising one or more regulatory elements operably linked to atransgene that results in selective expression in any target cell type,e.g., PV neurons in the CNS, over one or more non-target cell types, ornon-PV cells in the CNS. In some cases, each regulatory elementcomprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragmentor a combination thereof, or (iii) a sequence with at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to (i) or (ii). In some cases, sequenceidentity is determined using BLAST. In some cases, at least one of theregulatory elements is human derived. In some cases, at least one of theregulatory elements is derived from a non-human mammal. In some cases,the regulatory elements are non-naturally occurring. In some cases, theregulatory elements result in selective expression of the transgene inPV neurons that is greater than expression of the same transgene whenoperably linked to a non-selective regulatory element, as measured by aco-localization assay. In some cases, the non-selective regulatoryelement is a constitutive promoter. In some cases, the non-selectiveregulatory element is any one of CAG, EF1α, SV40, CMV, UBC, PGK, andCBA. In some instances, the regulatory elements result in selectiveexpression of the transgene in PV neurons at a level that is at least1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9fold, at least 10 fold, at least 15 fold, or at least 20 fold ascompared to selective expression of the transgene in PV neurons whenoperably linked to a non-selective regulatory element, as measured bythe co-localization assay. In some cases, the regulatory elements resultin selective expression in PV neurons that is at least 2%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% higher than expression in PVneurons when the transgene is operably linked to a non-selectiveregulatory element. In some cases, the regulatory elements result inselective expression in PV neurons that is about 1.5 times, 2 times, 2.5times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50times, or 100 times higher than expected for natural distribution of PVneurons in the CNS. In some cases, the co-localization assay is animmunohistochemical assay. In some cases, the immunohistochemical assaycomprises an anti-PV antibody. In some cases, the co-localization assayis performed as shown in Example 5 below. In some cases, the transgeneencodes an ion channel subunit, a neurotransmitter regulator, a DNAbinding domain, a gene editing protein, or a variant or a functionalfragment thereof. In some cases, the ion channel subunit is an alphasubunit or a beta subunit of a sodium ion channel or a subunit of apotassium ion channel. In some cases, the transgene comprises any one of(i) SEQ ID NOs: 37-43; (ii) a functional fragment thereof; or (iii) asequence having at least 80% sequence identity to (i) or (ii). In somecases, sequence identity is determined using BLAST. In some cases, thetransgene comprises (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, orKV3.3; (ii) a functional fragment thereof; or (iii) a sequence having atleast 80% sequence identity to (i) or (ii). In some cases, the transgeneis a neurotransmitter regulator that comprises (i) STXBP1, (ii) afunctional fragment thereof, or (iii) a sequence having at least 80%sequence identity to (i) or (ii). In some cases, the transgene comprisesa DNA binding protein that modulates expression of an endogenous gene.In some cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B,KV3.1, KV3.2, KV3.3, or STXBP1. In some cases, the transgene comprises aDNA binding protein that comprises a DNA binding domain of a DNA bindingprotein or a DNA cleaving protein (e.g., a nuclease, a restrictionenzyme, a recombinase, etc.) wherein the DNA cleaving domain or nucleasedomain has been deactivated, e.g., a nuclease-deactivated Cas (dCas), adeactivated transcription activator-like effector nuclease, or anuclease-deactivated zinc finger protein. In some cases, the transgenecomprises a DNA binding domain linked to a transcriptional modulatingdomain (e.g., a transcriptional activator or repressor domain). In somecases, the gene editing protein is a Cas protein. In some cases, theregulatory elements combined are less than 2.5 kb, less than 2 kb, lessthan 1.5 kb, less than 1 kb, or less than 500 bp in size. In some cases,the non-PV cells comprise one or more of non-PV cell types in the CNS.In some cases, the non-PV cells comprise one or more of excitatoryneurons, dopaminergic neurons, astrocytes, microglia, and motor neurons.In some cases, the nucleic acid cassette is a linear construct. In somecases, the nucleic acid cassette is a vector. In some cases, the vectoris a plasmid. In some cases, the vector is a viral vector. In somecases, the viral vector is an adeno-associated virus (AAV) vector. Insome cases, the AAV vector is AAV1, AAV8, AAV9, scAAV1, scAAV8, orscAAV9. In some cases, the viral vector is a lentiviral vector.

In one aspect, regulatory elements of any of the nucleic acid cassettesdisclosed herein contain less than 600 bp of contiguous sequence fromwithin 10 kb of the transcription start site of GAD2, GAD1, SYN1,NKX2.1, DLX1, DLX5/6, SST, PV, and/or VIP.

In one aspect, a method of treating a neurological disorder or conditionin a subject in need thereof comprises delivering a therapeuticallyeffective amount of any of the nucleic acid cassette disclosed herein.In some cases, the neurological disorder or condition is a psychiatricdisorder (e.g., schizophrenia, obsessive compulsive disorder, addiction,depression, anxiety, psychosis); an autism spectrum disorder (e.g.,Fragile X syndrome, Rett syndrome); epilepsy (e.g., chronic traumaticencephalopathy, generalized epilepsy with febrile seizures plus (GEFS+),epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy,tuberous sclerosis); or neurodegeneration (e.g., Alzheimer's disease,Parkinson's disease). In some cases, the neurological disorder orcondition is Dravet syndrome or Alzheimer's disease. In some cases, theneurological condition or disease is any seizure and/or epilepsy relatedcondition or disease wherein PV neurons are implicated.

In one aspect, a method of increasing selective expression of atransgene in PV neurons in CNS comprises contacting a cell with anucleic acid cassette disclosed herein.

In some aspects, the present disclosure contemplates a method oftargeting expression of any transgene to PV neurons in the CNS, themethod comprising operably linking one or more of PV neuron selectiveregulatory elements to a transgene. In some cases, each of theregulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) afunctional fragment or a combination thereof, or (iii) a sequence withat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to (i) or (ii). In somecases, sequence identity is determined using BLAST. In some cases, theregulatory elements result in selective expression of the transgene inPV neurons that is greater than expression of the same transgene whenoperably linked to a non-selective regulatory element, as measured by aco-localization assay. In some cases, the immunohistochemical assaycomprises an anti-PV antibody (e.g., as described in Example 5 below).In some cases, the non-selective regulatory element is a constitutivepromoter. In some cases, the non-selective regulatory element is any oneof CAG, EF1α, SV40, CMV, UBC, PGK, and CBA. In some cases, theregulatory elements result in selective expression of the transgene inPV neurons at a level that is at least 1.5 fold, at least 2 fold, atleast 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, atleast 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, atleast 15 fold, or at least 20 fold as compared to a non-selectiveregulatory element when operably linked to the transgene, as measured bya co-localization assay. In some cases, the regulatory elements resultin selective expression in PV neurons that is at least 2%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% higher than expression in PVneurons when the transgene is operably linked to a non-selectiveregulatory element. In some cases, the regulatory elements result inselective expression in PV neurons that is about 1.5 times, 2 times, 2.5times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50times, or 100 times higher than expected for natural distribution of PVneurons in CNS. In some cases, the transgene is any one of SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, STXBP1, a DNA binding protein, a geneediting protein, or a functional fragment thereof. In some cases, theregulatory elements and the transgene are in an AAV. In some cases, theAAV is AAV1, AAV8, AAV9, scAAV1, scAAV8, or scAAV9.

In another aspect, the present disclosure contemplates a method oftreating a neurological condition or disorder in a subject in needthereof, the method comprising contacting a cell with a nucleic acidcassette comprising: one or more regulatory elements operably linked toa transgene that result in selective expression of the transgene in PVneurons over one or more non-PV cells in CNS. In some cases, each of theregulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) afunctional fragment or a combination thereof, or (iii) a sequence withat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to (i) or (ii). In somecases, sequence identity is determined using BLAST. In some cases, thetransgene is a voltage-gated ion channel subunit, or a variant or afunctional fragment thereof. In some cases, the subunit is a betasubunit of a sodium ion channel. In some cases, the subunit is an alphasubunit of a sodium ion channel. In some cases, the subunit is of apotassium ion channel. In some cases, the transgene is any one of (i)SCN1A, SCN1B, SCN2B, KV3.1, or KV3.3; (ii) a functional fragmentthereof; or (iii) a sequence having at least 80% sequence identity to(i) or (ii). In some cases, the transgene is a DNA binding protein. Insome cases, the DNA binding protein modulates an endogenous gene. Insome cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B,KV3.1, KV3.3, or STXBP1. In some cases, the transgene is a DNA bindingprotein that comprises a DNA binding domain of a DNA binding protein ora DNA cleaving protein (e.g., a nuclease, a restriction enzyme, arecombinase, etc.) wherein the DNA cleaving domain or nuclease domainhas been deactivated, e.g., a nuclease-deactivated Cas (dCas), adeactivated transcription activator-like effector nuclease, or anuclease-deactivated zinc finger protein. In some cases, the transgenecomprises a DNA binding domain linked to a transcriptional modulatingdomain (e.g., a transcriptional activator or repressor domain). In somecases, the transgene is a gene editing protein. In some cases, the geneediting protein is a Cas protein, e.g., Cas9. In some cases, theneurological condition or disorder is associated with ahaploinsufficiency or a mutation in any of SCN1A, SNC2A, SNC8A, SCN1B,SCN2B, KV3.1, KV3.3, or STXBP1. In some cases, the neurologicalcondition or disorder is epilepsy, neurodegeneration, tauopathy, orneuronal hypoexcitability. In some cases, the neurological condition ordisorder is Dravet syndrome. In some cases, the neurological conditionor disorder is Alzheimer's disease. In some cases, the neurologicalcondition or disease is a psychiatric disorder (e.g., schizophrenia,obsessive compulsive disorder, addiction, depression, anxiety,psychosis); an autism spectrum disorder (e.g., Fragile X syndrome, Rettsyndrome); epilepsy (e.g., chronic traumatic encephalopathy, generalizedepilepsy with febrile seizures plus (GEFS+), epileptic encephalopathy,temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); orneurodegeneration (e.g., Alzheimer's disease, Parkinson's disease). Insome cases, the neurological condition or disease is any seizure and/orepilepsy related condition or disease wherein PV neurons are implicated.In some cases, the regulatory elements of this disclosure result inselective expression of the transgene in PV neurons that is greater thanexpression of the same transgene when operably linked to a non-selectiveregulatory element, as measured by a co-localization assay. In somecases, the non-selective regulatory element is a constitutive promoter.In some cases, the non-selective regulatory element is any one of CAG,EF1α, SV40, CMV, UBC, PGK, and CBA. In some cases, the regulatoryelements result in selective expression in PV neurons at a level that isat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 15 fold, or at least 20 fold ascompared to a non-selective regulatory element when operably linked tothe transgene, as measured by a co-localization assay. In some cases,the regulatory elements result in selective expression in PV neuronsthat is at least 2%, at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% higher than expression in PV neurons when the transgene is operablylinked to a non-selective regulatory element. In some cases, theregulatory elements result in selective expression in PV neurons that isabout 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8times, 8.5 times, 9 times, 9.5 times, 10 times, 15 times, 20 times, 25times, 30 times, 40 times, 50 times, or 100 times higher than expectedfor natural distribution of PV neurons in CNS. In some cases, thenucleic acid cassette is in an AAV. In some cases, the AAV is AAV1,AAV8, AAV9, scAAV1, scAAV8, or scAAV9.

In one aspect, the present disclosure provides a method of treatingDravet syndrome, comprising contacting a cell with an AAV comprising atransgene, wherein the transgene is any one of (i) SCN1A, SNC2A, SNC8A,SCN1B, SCN2B, or a DNA binding protein, (ii) a functional fragmentthereof, or (iii) a sequence having at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to (i) or (ii). In some cases, sequence identity is measuredusing BLAST. In some cases, the DNA binding protein modulates anendogenous gene. In some cases, the DNA binding protein is atranscriptional modulator. In some cases, the transgene is a DNA bindingprotein that comprises a DNA binding domain of a DNA binding protein ora DNA cleaving protein (e.g., a nuclease, a restriction enzyme, arecombinase, etc.) wherein the DNA cleaving domain or nuclease domainhas been deactivated, e.g., a nuclease-deactivated Cas (dCas), adeactivated transcription activator-like effector nuclease, or anuclease-deactivated zinc finger protein. In some cases, the DNA bindingdomain is linked to a transcriptional modulating domain (e.g., atranscriptional activator or repressor domain). In some cases, thetransgene comprises a gene editing protein, e.g., a Cas protein, Cas9.In some cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, orSCN2B. In some cases, the AAV further comprises one or more PV neuronselective regulatory elements or one or more regulatory elementsdisclosed herein operably linked to the transgene. In some cases, eachof the regulatory elements independently comprises (i) a sequence of SEQID NOs: 1-32, (ii) a functional fragment or a combination thereof, or(iii) a sequence with at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity to(i) or (ii).

In another aspect, the present disclosure provides a method of treatingAlzheimer's disease, comprising contacting a cell with an AAV comprisinga transgene, wherein the transgene is any one of (i) SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, STXBP1, or a DNA binding protein;(ii) a functional fragment thereof; or (iii) a sequence having at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to (i) or (ii). In some cases,sequence identity is measured using BLAST. In some cases, the DNAbinding protein modulates an endogenous gene. In some cases, theendogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, orSTXBP1. In some cases, the transgene is a DNA binding protein comprisinga transcriptional modulator. In some cases, the transgene is a DNAbinding protein that comprises a DNA binding domain of a DNA bindingprotein or a DNA cleaving protein (e.g., a nuclease, a restrictionenzyme, a recombinase, etc.) wherein the DNA cleaving domain or nucleasedomain has been deactivated, e.g., a nuclease-deactivated Cas (dCas), adeactivated transcription activator-like effector nuclease, or anuclease-deactivated zinc finger protein. In some cases, the DNA bindingdomain is linked to a transcriptional modulating domain (e.g., atranscriptional activator or repressor domain). In some cases, thetransgene comprises a gene editing protein, e.g., a Cas protein, Cas9.In some cases, the AAV further comprises one or more PV neuron selectiveregulatory elements or one or more regulatory elements disclosed hereinoperably linked to the transgene. In some cases, each of the regulatoryelements independently comprises each of the regulatory elementsindependently comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) afunctional fragment or a combination thereof, or (iii) a sequence withat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to (i) or (ii).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the frequency of seizures (seizures per 12 hrinterval) in SCN1A heterozygous mice after treatment with a recombinantAAVDJ vector comprising either SCN1B or eGFP operably linked to aregulatory element comprising a sequence of SEQ ID NO: 32. The graphillustrates the mean values at each day of recording with error barsrepresenting the standard error of the mean.

FIG. 2 illustrates high gamma power (50-100 Hz) of different mice:wild-type control (WT), untreated transgenic APP/PS1 mice (APP/PS1), ortransgenic APP/PS1 mice treated with rAAV comprising SCN1B operablylinked to a regulatory element comprising a sequence of SEQ ID NO: 32(APP/PS1+SCN1B).

FIG. 3A illustrates immunofluorescence co-localization assay of CNScells from pups following neonatal systemic injections of AAV9comprising eGFP transgene operably linked to a regulatory elementcomprising a sequence of SEQ ID NO: 1 or SEQ ID NO: 8. AAV9 comprisingeGFP transgene operably linked to CAG was used as a control. Lower rowimages illustrate eGFP+ cells. Middle row images illustrate PV+ cells,which were stained with an anti-PV antibody. Top row images (merge)illustrate an overlay of PV+, eGFP+ fluorescence (with representativeeGFP+ and PV+ cells which are shown as white or light grey cellsindicated by arrowheads) and DAPI+.

FIG. 3B illustrates the quantification of immunofluorescenceco-localization studies illustrated in FIG. 3A, wherein selectiveexpression in PV cells is expressed as the percentage of eGFP+ cellsthat were also PV+ in comparison to the CAG control, as measured by theimmunofluorescence co-localization assay.

FIG. 4A illustrates immunofluorescence co-localization assay of CNScells from adult mice following systemic injections of AAV9 comprisingeGFP transgene operably linked to a regulatory element comprising asequence of SEQ ID NO: 1 or SEQ ID NO: 8. AAV9 comprising eGFP transgeneoperably linked to EF1α was used as a control. Lower row imagesillustrate eGFP+ cells. Middle row images illustrate PV+ cells, whichwere stained with an anti-PV antibody. Top row images (merge) illustratean overlay of PV+ eGFP+ fluorescence (with representative eGFP+ and PV+cells, or the white or light grey cells, indicated by arrowheads) andDAPI+.

FIG. 4B illustrates the quantification of immunofluorescenceco-localization studies illustrated in FIG. 4A, wherein selectiveexpression in PV cells is expressed as the percentage of eGFP+ cellsthat were also PV+ in comparison to the EF1α control, as measured by theimmunofluorescence co-localization assay.

FIGS. 5A-5F illustrate immunofluorescence co-localization assay of CNScells from adult mice following direct CNS injections of AAVDJcomprising eGFP transgene operably linked to a regulatory elementcomprising a sequence of SEQ ID NOs: 2-22. Lower row images illustrateeGFP+ cells. Middle row images illustrate PV cells that were stainedwith an anti-PV antibody. Top row images (merge) illustrate an overlayof PV+, eGFP+ fluorescence (with representative eGFP+ and PV+ cells, orthe white or light grey cells, indicated by arrowheads) and DAPI+. FIG.5A illustrates the immunofluorescence co-localization assay performedwith AAVDJ comprising one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,and SEQ ID NO: 5 operably linked to eGFP. FIG. 5B illustrates theimmunofluorescence co-localization assay performed with AAVDJ comprisingone of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9 operably linked toeGFP. FIG. 5C illustrates the immunofluorescence co-localization assayperformed with AAVDJ comprising one of SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, and SEQ ID NO: 13 operably linked to eGFP. FIG. 5Dillustrates the immunofluorescence co-localization assay performed withAAVDJ comprising one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, andSEQ ID NO: 17 operably linked to eGFP. FIG. 5E illustrates theimmunofluorescence co-localization assay performed with AAVDJ comprisingone of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21operably linked to eGFP. FIG. 5F illustrates the immunofluorescenceco-localization assay performed with AAVDJ comprising SEQ ID NO: 22 orSEQ ID NO: 34 operably linked to eGFP, wherein SEQ ID NO: 34 is apreviously characterized non-selective regulatory element and was usedas a control for comparison.

FIG. 6 illustrates the quantification of immunofluorescenceco-localization studies illustrated in FIGS. 5A-5F, wherein selectiveexpression in PV cells is expressed as the percentage of eGFP+ cellsthat were also PV+ in comparison to SEQ ID NO: 34, as measured by theimmunofluorescence co-localization assay.

FIG. 7 illustrates a schematic of an example of an expression cassettecontaining REs of this disclosure, e.g., an enhancer, a promoter, andstability elements. REs can be located upstream and/or downstream of atransgene in an expression cassette, which can be a plasmid, vector, ora viral vector.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure contemplates compositions and methods of usingsuch compositions in gene therapy to treat a disease or conditionassociated with the central nervous system (CNS), e.g., Dravet syndrome,Alzheimer's disease, epilepsy, and/or seizures.

Gene therapy can replace, modify, delete, or add a gene or a specificnucleic acid sequence, such as an expression cassette, to impart atherapeutic effect in a cell. In some cases, gene therapy is used todeliver an expression cassette into a cell that produces or results in atherapeutic effect. In some cases, a virus, such as AAV, comprising aviral vector that comprises an expression cassette can be used todeliver a transgene into a cell. The expression cassette can contain atransgene that provides a therapeutic effect when expressed in a cell.

One challenge in gene therapy is ensuring that the transgene isexpressed in an appropriate cell type of interest, or the target celltype, to effect or target gene expression. Traditional methods fortargeting gene therapy have often relied on delivery methods and/orvehicles (e.g., varying the viruses used or capsid sequences ofviruses). In addition to targeting, or selective expression, of anexpression cassette in the target cell type over one or more non-targetcell types, another challenge in the field is increasing geneexpression, especially when the gene is large, in a target cell type ortissue to exert a therapeutic effect.

The present disclosure provides a plurality of regulatory elements,which are non-coding nucleotide sequences, that can be operably linkedto any transgene to increase or to improve selectivity of the transgeneexpression in the CNS, e.g., in PV neurons. By increasing selectivity ofgene expression using one or more regulatory elements disclosed herein,one can improve the efficacy of a gene therapy, decrease the effectivedose needed to result in a therapeutic effect, minimize adverse effectsor off-target effect, and/or increase patient safety and/or tolerance.

In one aspect, one or more regulatory elements can be operably linked toany transgene in an expression cassette to modulate gene expression in acell, such as targeting expression of the transgene in a target celltype or tissue (e.g., PV cells) over one or more non-target cell type ortissue (e.g., non-PV CNS cell-types). In some cases, targetingexpression of the transgene in a target cell type or tissue includesincreased gene expression in the target cell type or tissue. One or moreregulatory elements operably linked to a transgene can be part of anexpression cassette, which can be a linear or a circular construct, aplasmid, a vector, a viral vector, e.g., a vector of an adeno-associatedvirus (AAV). Such expression cassette can be adapted for gene therapy ordelivery into a subject (e.g., a human, a patient, or a mammal). In somecases, operably linking one or more regulatory elements to a generesults in targeted expression of the gene in a target tissue or celltype in the CNS, such as a parvalbumin (PV) neuron. In some cases, oneor more regulatory elements (e.g., SEQ ID NOs: 1-32, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 90%, at least 95%, or at least 99% sequence identity thereto)increase selectivity of gene expression in a target tissue or cell typein the CNS, such as PV neurons. In some cases, a gene therapy comprisesone or more regulatory elements disclosed herein, wherein the regulatoryelements are operably linked to a transgene and drive selectiveexpression of the transgene in PV neurons.

In some cases, selective expression of a gene in PV neurons is used totreat a disease or condition associated with a haploinsufficiency and/ora genetic defect in an endogenous gene, wherein the genetic defect canbe a mutation in the gene or dysregulation of the gene. Such geneticdefect can result in a reduced level of the gene product and/or a geneproduct with impaired function and/or activity. In some cases, anexpression cassette comprises a gene, a subunit, a variant or afunctional fragment thereof, wherein gene expression from the expressioncassette is used to treat the disease or condition associated with thegenetic defect, impaired function and/or activity, and/or dysregulationof the endogenous gene. In some cases, the disease or condition isDravet syndrome, Alzheimer's disease, epilepsy, neurodegeneration,tauopathy, neuronal hypoexcitability and/or seizures.

In some cases, the transgene is an ion channel or a neurotransmitterregulator, a DNA binding protein, or a subunit, variant, or functionalfragment thereof. In some cases, the transgene is a sodium ion channelalpha subunit, sodium ion channel beta subunit, or a variant orfunctional fragment thereof. In some cases, the transgene is a potassiumion channel or a subunit thereof. In some cases, the transgene is SCN1A,SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, a DNA bindingprotein (e.g., a DNA binding protein that modulates expression of anendogenous gene), or a variant or functional fragment thereof. In somecases, the transgene comprises a sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to any one of SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, a DNA binding protein,or a variant or functional fragment thereof. In some cases, thetransgene is a DNA binding protein that modulates expression of anendogenous gene, such as any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B,KV3.1, KV3.2, KV3.3, and STXBP1.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within one or more than one standarddeviation, per the practice in the art. Alternatively, “about” can meana range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1%) of agiven value.

The terms “determining”, “measuring”, “evaluating”, “assessing”,“assaying”, “analyzing”, and their grammatical equivalents can be usedinterchangeably herein to refer to any form of measurement, and includedetermining if an element is present or not (for example, detection).These terms can include both quantitative and/or qualitativedeterminations. Assessing may be relative or absolute.

The term “expression” refers to the process by which a nucleic acidsequence or a polynucleotide is transcribed from a DNA template (such asinto mRNA or other RNA transcript) and/or the process by which atranscribed mRNA is subsequently translated into peptides, polypeptides,or proteins. Transcripts and encoded polypeptides may be collectivelyreferred to as “gene product.” If the polynucleotide is derived fromgenomic DNA, expression may include splicing of the mRNA in a eukaryoticcell.

As used herein, “operably linked”, “operable linkage”, “operativelylinked”, or grammatical equivalents thereof refer to juxtaposition ofgenetic elements, e.g., a promoter, an enhancer, a polyadenylationsequence, etc., wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a regulatoryelement, which can comprise promoter and/or enhancer sequences, isoperatively linked to a coding region if the regulatory element helpsinitiate transcription of the coding sequence. There may be interveningresidues between the regulatory element and coding region so long asthis functional relationship is maintained.

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Examples of vectors include plasmids, viral vectors, liposomes, andother gene delivery vehicles. The vector generally comprises geneticelements, e.g., regulatory elements, operatively linked to a gene tofacilitate expression of the gene in a target. The combination ofregulatory elements and a gene or genes to which they are operablylinked for expression is referred to as an “expression cassette”.

The term “AAV” is an abbreviation for adeno-associated virus, and may beused to refer to the virus itself or a derivative thereof. The termcovers all serotypes, subtypes, and both naturally occurring andrecombinant forms, except where required otherwise. The abbreviation“rAAV” refers to recombinant adeno-associated virus, also referred to asa recombinant AAV vector (or “rAAV vector”). The term “AAV” includesAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV,equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomicsequences of various serotypes of AAV, as well as the sequences of thenative terminal repeats (TRs), Rep proteins, and capsid subunits areknown in the art. Such sequences may be found in the literature or inpublic databases such as GenBank. An “rAAV vector” as used herein refersto an AAV vector comprising a polynucleotide sequence not of AAV origin(i.e., a polynucleotide heterologous to AAV), typically a sequence ofinterest for the genetic transformation of a cell. In general, theheterologous polynucleotide is flanked by at least one, and generally bytwo, AAV inverted terminal repeat sequences (ITRs). The term rAAV vectorencompasses both rAAV vector particles and rAAV vector plasmids. An rAAVvector may either be single-stranded (ssAAV) or self-complementary(scAAV). An “AAV virus” or “AAV viral particle” or “rAAV vectorparticle” refers to a viral particle composed of at least one AAV capsidprotein and an encapsidated polynucleotide rAAV vector. If the particlecomprises a heterologous polynucleotide (i.e., a polynucleotide otherthan a wild-type AAV genome such as a transgene to be delivered to amammalian cell), it is typically referred to as an “rAAV vectorparticle” or simply an “rAAV vector”. Thus, production of rAAV particlenecessarily includes production of rAAV vector, as such a vector iscontained within an rAAV particle.

As used herein, the terms “treat”, “treatment”, “therapy” and the likerefer to obtaining a desired pharmacologic and/or physiologic effect,including, but not limited to, alleviating, delaying or slowing theprogression, reducing the effects or symptoms, preventing onset,inhibiting, ameliorating the onset of a diseases or disorder, obtaininga beneficial or desired result with respect to a disease, disorder, ormedical condition, such as a therapeutic benefit and/or a prophylacticbenefit. “Treatment,” as used herein, covers any treatment of a diseasein a mammal, particularly in a human, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease or at risk of acquiring the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, i.e., causing regression ofthe disease. A therapeutic benefit includes eradication or ameliorationof the underlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder. In somecases, for prophylactic benefit, the compositions are administered to asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease, eventhough a diagnosis of this disease may not have been made. The methodsof the present disclosure may be used with any mammal. In some cases,the treatment can result in a decrease or cessation of symptoms (e.g., areduction in the frequency or duration of seizures). A prophylacticeffect includes delaying or eliminating the appearance of a disease orcondition, delaying or eliminating the onset of symptoms of a disease orcondition, slowing, halting, or reversing the progression of a diseaseor condition, or any combination thereof.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a composition described herein that is sufficient toaffect the intended application, including but not limited to diseasetreatment, as defined below. The therapeutically effective amount mayvary depending upon the intended treatment application (in vivo), or thesubject and disease condition being treated, e.g., the weight and age ofthe subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in a target cell. The specific dose willvary depending on the particular composition chosen, the dosing regimento be followed, whether it is administered in combination with othercompounds, timing of administration, the tissue to which it isadministered, and the physical delivery system in which it is carried.

A “fragment” of a nucleotide or peptide sequence is meant to refer to asequence that is less than that believed to be the “full-length”sequence.

A “variant” of a molecule refers to allelic variations of suchsequences, that is, a sequence substantially similar in structure andbiological activity to either the entire molecule, or to a fragmentthereof.

The term “functional fragment” is intended to include the “fragments”,“variants”, “analogues”, or “chemical derivatives” of a molecule.

A “functional fragment” of a DNA or protein sequence possesses at leasta biologically active fragment of the sequence, which refers to afragment that retains a biological activity (either functional orstructural) that is substantially similar to a biological activity ofthe full-length DNA or protein sequence. A biological activity of a DNAsequence can be its ability to influence expression in a manner known tobe attributed to the full-length sequence. For example, a functionalfragment of a regulatory element will retain the ability to influencetranscription as the full-length RE.

The terms “subject” and “individual” are used interchangeably herein torefer to a vertebrate, preferably a mammal, more preferably a human.“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in human therapeutics,veterinary applications, and/or preclinical studies in animal models ofa disease or condition. In some case, the subject is a mammal, and insome cases, the subject is human.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. For example, an in vitro assay encompasses any assay runoutside of a subject. In vitro assays encompass cell-based assays inwhich cells alive or dead are employed. In vitro assays also encompass acell-free assay in which no intact cells are employed.

Sequence comparisons, such as for the purpose of assessing identities,mutations, or where one or more positions of a test sequence fallrelative to one or more specified positions of a reference sequence, maybe performed by any suitable alignment algorithm, including but notlimited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needlealigner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionallywith default settings), the BLAST algorithm (see, e.g., the BLASTalignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionallywith default settings), and the Smith-Waterman algorithm (see, e.g., theEMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/,optionally with default settings). Optimal alignment may be assessedusing any suitable parameters of a chosen algorithm, including defaultparameters.

In general, “sequence identity” or “sequence homology”, which can beused interchangeably, refer to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Typically, techniques fordetermining sequence identity include determining the nucleotidesequence of a polynucleotide and/or determining the amino acid sequenceencoded thereby, and comparing these sequences to a second nucleotide oramino acid sequence. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity”, alsoreferred to as “percent homology”. The percent identity to a referencesequence (e.g., nucleic acid or amino acid sequences), which may be asequence within a longer molecule (e.g., polynucleotide or polypeptide),may be calculated as the number of exact matches between two optimallyaligned sequences divided by the length of the reference sequence andmultiplied by 100. Percent identity may also be determined, for example,by comparing sequence information using the advanced BLAST computerprogram, including version 2.2.9, available from the National Institutesof Health. The BLAST program is based on the alignment method of Karlinand Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and asdiscussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlinand Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); andAltschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, theBLAST program defines identity as the number of identical alignedsymbols (i.e., nucleotides or amino acids), divided by the total numberof symbols in the shorter of the two sequences. The program may be usedto determine percent identity over the entire length of the sequencesbeing compared. Default parameters are provided to optimize searcheswith short query sequences, for example, with the blastp program. Theprogram also allows use of an SEG filter to mask-off segments of thequery sequences as determined by the SEG program of Wootton andFederhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desireddegrees of sequence identity are approximately 80% to 100% and integervalues there between. Typically, the percent identities between adisclosed sequence and a claimed sequence are at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99%. Ingeneral, an exact match indicates 100% identity over the length of thereference sequence. In some cases, reference to percent sequenceidentity refers to sequence identity as measured using BLAST (BasicLocal Alignment Search Tool). In other cases, ClustalW can be used formultiple sequence alignment.

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ, conventional techniques of molecular biology,microbiology, and recombinant DNA technology, which are within theknowledge of those of skill of the art.

Regulatory Elements

Regulatory elements are nucleic acid sequences or genetic elements whichare capable of influencing (e.g., increasing or decreasing) expressionof a gene and/or confer selective expression of a gene (e.g., a reportergene such as eGFP, a transgene, or a therapeutic gene) in a particulartissue or cell type of interest. In some cases, a regulatory element canbe a transgene, an intron, a promoter, an enhancer, UTR, insulator, arepressor, an inverted terminal repeat (ITR) sequence, a long terminalrepeat sequence (LTR), stability element, posttranslational responseelement, or a polyA sequence, or a combination thereof. In some cases,the regulatory element is a promoter or an enhancer, or a combinationthereof. In some cases, the regulatory element is derived from a humansequence.

In some cases, the cell type of interest is a PV neuron. Regulatoryelements can function at the DNA and/or the RNA level. Regulatoryelements can function to modulate gene expression selectivity in a celltype of interest. Regulatory elements can function to modulate geneexpression at the transcriptional phase, post-transcriptional phase, orat the translational phase of gene expression. Regulatory elementsinclude, but are not limited to, promoter, enhancer, repressor,silencer, and insulator sequences. At the RNA level, regulation canoccur at the level of translation (e.g., stability elements thatstabilize mRNA for translation), RNA cleavage, RNA splicing, and/ortranscriptional termination. In some cases, regulatory elements canrecruit transcriptional factors to a coding region that increase geneexpression selectivity in a cell type of interest. In some cases,regulatory elements can increase the rate at which RNA transcripts areproduced, increase the stability of RNA produced, and/or increase therate of protein synthesis from RNA transcripts. In some cases,regulatory elements can prevent RNA degradation and/or increase itsstability to facilitate protein synthesis. In some cases, regulatoryelements suppress transcription and/or translation processes inoff-target cell-types. In some cases, off-target cell-types include, butare not limited to, excitatory neurons, non-PV CNS cell-types, andnon-neuronal CNS cell types.

Various assays including, but not limited to, DNAase hypersensitivity,ATAC-Seq, and ChIP-Seq can be used to identify putative non-codingregulatory elements (REs). The enzymatic reaction in each of theseassays preferentially targets open/accessible chromatin states, a statewhich is thought to be predictive of regulatory elements. To discovercell-type selective regulatory elements, one can assay for openchromatin sequence for target cell-type of interest (e.g., parvalbuminneurons) and compare that to open chromatin sequences for non-targetcell types (e.g., excitatory neurons). Additional filters can be appliedto further refine target selection, including proximity to a cell-typeselective gene, species conservation, and/or sequence motifs, such astranscription factor binding sites. DNA sequences that are uniquelyidentified in the target cell type can be synthesized and cloned into anexpression vector. The selectivity of a regulatory element can bedetermined using immunohistochemical methods to quantify co-localizationto known cell-type selective proteins.

For example, one method of isolating a cell-type selective regulatoryelement includes isolating nuclei from a brain tissue or cell type ofinterest from an animal model, which can be achieved by using anaffinity purification method that isolates the tissue or cell type ofinterest (e.g., using beads coated to an anti-PV antibody for isolatingPV neurons), using high-throughput natural priming and DNA synthesis togenerate a pool of sequences from open chromatin regions in the nuclei,sequencing the pool of sequences to identify putative sequences thatdrive gene expression in the tissue or cell type of interest, andverifying selective expression in a reporter system in a cell line invitro and/or in an animal model.

Another method for identifying candidate regulatory elements that areselective in a tissue or cell type of interest include usingR26-CAG-LSL-Sunl-sfGFP-Myc knockin mouse for harvesting the tissue orcell type of interest, isolating GFP+/Myc+ nuclei from the mouseneocortex of this strain using affinity purification, e.g., usinganti-GFP or anti-Myc antibodies and protein G-coated magnetic beads toisolate nuclei from the neocortex. Nuclear RNA from purified nuclei orwhole neocortical nuclei can be converted to cDNA and amplified with theNugen Ovation RNA-seq System V2 (Nugen 7102), followed by sequencingusing the Illumina HiSeq 2500. Genomic DNA from purified nuclei can befragmented and used to make MethylC-seq libraries, which can besequenced using the Illumina HiSeq 2000. To generate an ATAC-seqlibrary, nuclei bound to beads are transposed using Tn5 transposase(Illumina FC-121-1030). After 9-12 cycles of PCR amplification,libraries are sequenced using an Illumina HiSeq 2500. To generate aChIP-seq library, excitatory neuron nuclei can be digested tomononucleosomes using micrococcal nuclease, followed by salt extractionof chromatin, and native ChIP and library construction, which can besequenced on an Illumina HiSeq 2500. After sequencing these libraries,the sequences are mapped to identify, for example, correlation incell-type-specific hypo-methylation in CG-rich regions, histonemodifications, transcriptional factor binding sites, and patternsassociated with highly expressed transcriptional factors. Overlappingfeatures and correlations from multiple assays and/or librariesdescribed above provide evidence for identifying candidate sequenceswithin such genomic regions as potential regulatory elements associatedwith selective expression and/or high expression in the cells isolatedfrom the neocortex. For example, a genomic region characterized by astrong overlap between hypomethylation detected in the methy 1C-seqlibrary, ChIP assay, and an enrichment in transcription factor bindingmotifs in the same region provide convergent data that indicate thegenomic region contains a sequence of a putative regulatory elementselective for the tissue or cell type isolated. As another example, toidentify candidate PV neuron selective regulatory elements, one canisolate PV neurons and purify nuclei from the isolated PV cells so thatgenomic sequences that are identified as active in multiple sequencingassays described above have a high likelihood of being PV cell-selectiveregulatory elements, e.g., a genomic region that is identified as activein an ATAC-seq assay (corresponding to regions of open chromatin),active in RNA-seq (indicative of active gene expression and low DNAmethylation patterns in the region), and active in methylC-seq assay(which generates single-base resolution methylome maps from a cell typeof interest).

Once candidate genomic regions are identified as selectively active in acell type of interest, sequences within the region can be generatedusing PCR methods and tested in additional assays in vitro and/or invivo to validate tissue or cell type selectivity of the sequences. Suchvalidation assays include immunohistochemical co-localization assay,wherein an antibody or any detectable marker is used to label the celltype of interest and a second detectable marker, e.g., a fluorescenttransgene, is operably linked to the putative regulatory elements.Expression cassettes comprising such elements are delivered into cellsin vitro and/or in vivo. Selective expression driven by one or moreputative regulatory elements can be validated by measuring the overlapbetween the cell type of interest (as measured by the detectable signalor fluorescence from its labeled marker, e.g., an anti-PV antibody) andthe second detectable marker corresponding to expression of thetransgene (e.g., eGFP or RFP) operably linked to the regulatoryelements. An overlap in the signals from both detectable markersindicates cell-type selectivity in the labeled cell type if the amountof overlap observed is higher than the overlap observed when theregulatory elements are replaced with a control, such as CAG, EF1α, aconstitutive promoter (e.g., SV40, CMV, UBC, PGK, and CBA), anon-selective regulatory element, or a previously characterizednon-selective regulatory element. Various mouse strains adapted forexpressing a detectable marker in a cell type of interest allowsvalidation of cell type selectivity of a regulatory element in vivo. Forexample, a number of mouse lines that express Cre in a particular celltype can be used because cell-type selective Cre expression can driveCre-induced expression of a fluorescent protein, such as RFP, in a celltype of interest. Labeling such cell type of interest in vivo allows oneto determine level of cell-type selective expression that is associatedwith a putative regulatory element operably linked to a fluorescent orreporter transgene in the same mouse. Similar to the co-localizationassay, an overlap of the signals from both markers that exceeds theoverlap detected for CAG, EF1α, a constitutive promoter (e.g., SV40,CMV, UBC, PGK, and CBA), or a non-selective regulatory element isindicative of cell type selectivity for the regulatory elements tested.In some cases, the mouse strain used is B6 PV-Cre mouse (JacksonLaboratory), which is a B6 PV-Cre knock-in mouse that expresses Crerecombinase in parvalbumin-expressing neurons (e.g., interneurons in thebrain and proprioceptive afferent sensory neurons in the dorsal rootganglia), without disrupting endogenous Pvalb expression.

Upon validation of cell type selectivity of a regulatory element for aparticular cell type, sequences of such regulatory elements can bevaried using various mutagenesis methods, e.g., error-prone PCR methods,to improve its selectivity. In some cases, two or more regulatoryelements having cell selectivity can be combined. In some cases,combined regulatory elements exhibit enhanced cell-type selectivity indriving gene expression in the cell type of interest. In some instances,such regulatory elements are truncated one or more bases at a time todetermine the minimal amount of sequence that retains its cell typeselectivity. Smaller regulatory elements that retain cell typeselectivity are helpful for making gene therapy comprising a largetransgene, or where the cloning capacity of a vector or plasmid islimited in view of the size of a transgene that one wishes to deliverusing gene therapy.

The present disclosure provides a plurality of nucleotide sequences thatare regulatory elements. In some cases, any one or more of theregulatory elements disclosed herein result in increased selectivity ingene expression in a parvalbumin cell. In some cases, regulatoryelements disclosed herein are PV-cell-selective. In some cases, PV cellselective regulatory elements are associated with selective geneexpression in PV cells more than expression in non-PV CNS cell-types. Insome cases, PV cell selective regulatory elements as associated withreduced gene expression in non-PV CNS cell types.

Non-limiting examples of regulatory elements include SEQ ID NOs: 1-32,as provided in TABLE 1 below.

TABLE 1 List of nucleic acid sequences disclosed herein. Source/ SEQ IDGenomic NO: Nucleic Acid Sequence Location  1GGAGGAAGCCATCAACTAAACTACAATGACTGTAAGATACAAA Human;ATTGGGAATGGTAACATATTTTGAAGTTCTGTTGACATAAAGAA hg19: chr2:TCATGATATTAATGCCCATGGAAATGAAAGGGCGATCAACACT 171621900-ATGGTTTGAAAAGGGGGAAATTGTAGAGCACAGATGTGTTCGT 171622580GTGGCAGTGTGCTGTCTCTAGCAATACTCAGAGAAGAGAGAGAACAATGAAATTCTGATTGGCCCCAGTGTGAGCCCAGATGAGGTTCAGCTGCCAACTTTCTCTTTCACATCTTATGAAAGTCATTTAAGCACAACTAACTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTTGCCCAGGACAGAGTGCAGTAGTGACTCAATCTCGGCTCACTGCAGCCTCCACCTCCTAGGCTCAAACGGTCCTCCTGCATCAGCCTCCCAAGTAGCTGGAATTACAGGAGTGGCCCACCATGCCCAGCTAATTTTTGTATTTTTAATAGATACGGGGGTTTCACCATATCACCCAGGCTGGTCTCGAACTCCTGGCCTCAAGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTCAGCCACTATGCCCAACCCGACCAACCTTTTTTAAAATAAATATTTAAAAAATT GGTATTTCACATATATACTAGT  2AGTTTGGACAAGAACTATAGTTCTAGCTTTCTCTGGGTCTCCAC Mouse;CTTGCAGAGAATGCAGCTTTCATTATCTCATGAGCCAAACTCTC mm10: chr2:ATCATCTCTTTCCATATATCTGTCGGTGCTCTTCCATGAGTACTC 36053858-TAACACACACAGAAGGAGCACTTACACAGGCTGTTGTTTTTCTC 36054359TTATTATCATAGCTGTTGTTCAGACATGTGCATTCTGTTCTTGTTGCTTCAATGCTAAAGGAGTCTCAGGATATGAGAACTGTACCAGCCGAGGCATCAGGAAACATGGGTGGAAATTCCCACAGTACTATTTGTTCACTGTGTGACCTTGGGCCAGTCACATCCCTTTCCTGAGGCTTCGATTCCCCAAGCTATAAAAGAAGCATCTCTTAACCTTTTTTTAGGTCATGAGTCAGGCCCAGCACACTCTCAGGGAGACTCATGAGAGTACAGATCATTTCCCATAGAAAAACCATAGTTTTATATC CAGAGGCTTTTCTGTAAG  3GGTTCCAGTTCAGAGGCAGAGCATTTGGGGTTCCCAGTCAGGA Mouse;GCTTTCCTCTCTCCGCTCCTTAGTTTCCTCTCTTTAAAAAAAAAT chr2:GGGTGATAGTATAGAAAGGAAGCTCTGGGCTCGGGGACCAGGG 36,091,144-CCCTGGGATCCCCGCTCCCAGCCACTCGCTCCTGACCCTTCCAG 36,091,966GGACAAGCTCCCCCCCACCCCGTCCTTTCCAGGCTGCCACTAGAAGAGATGGGGACGCGTGGTCAGCCGCTTCTGTCGCCCCCCAGGGAACGGTCTCACGCTGGAGGGGGCAGTGCCCTCGGAACAGGACAGTCAGCCCAAGCCAGCCAAGCGCGCGCGGACGTCCTTCACCGCAGAGCAATTGCAGGTACCCCGGGCAAGCCCCGAAGCGTGTGGGCGGGGCTTCGGAGTGGGCGTGGTTGTTCGGGACTTGTGACTCCGCCCCTTGTGCGGGGACCCGCGTGAGGCCGCTCCAAGGATGAAGCTGCCTGGGGCGTGGCCTCGGACCCTGAGCCTCTGATTGGGCGGAGGTCTCAGGGCCCTTCTGCGCCCCACAGGTTATGCAGGCGCAGTTCGCGCAGGACAACAACCCGGACGCGCAGACGCTGCAGAAGCTGGCGGACATGACGGGCCTCAGTCGCAGGGTCATCCAGGTGGGGCTCCGGGGTCTCGGCCTTCAGGTCTAGGGTGAACCTTAGGGAAGCGCTGAAGCTCGTAGTGGTACGGATGGTCGCGCGTGCACGTGGCCGCCCCTCTCCAGTGTGGCCTAAGGACCCCAGTCGGCACGGGTTGACCCTTTTCCTTGATTACTGAGAGTGCAGAGGCTGT  4TGGTGGGAAGACATGTCCAGGGAAGAAATGGCCTCCAGAGGCC Mouse;TGAGGTGGGGAAATGCTGGAGGTGGAGAGAGGAACAACTGACT chr2:GAAAATGAGCTTCCACTGTGGCTTAGTAGCCTATACCAAGTCTA 36,095,396-GAGTATAGGGTAGGAGAAGATTAGGAAAGCGATGGGTCTGAGA 36,096,028ATGATGTGGCCTGTTGACTTTTGTAAACCCAAAGCACCTTGGACTAAACCCTATGAACAGTGTGGTGCCACCAAAGACTATAATGAGCTCAGGGAACAGAATTCTGTGTGCATGGTGATTTTTTTTTTTTTTTTCTGCTAACTGCAGTCTGGGTGATGCATTGACAAACCAATCCTGGAAAGTAAGAGGCAAGGGCAGCTGGGACGGTGAGAGGAGCCTGATGGGAACCAGGCCAAGCAGGGCAGCAGAGGCGATGAAGAGGATGTGGTGCATCCAGAGACTCACTTCATTAGCTGGAGGCACTGCTGGATAGGGTCTGAAGGTTCTGGTATCTGAGTTGGCGGGCTGGGTGAGTGGTGGCTCTGCTTCCTGAACAGTGTGTGCAAGAGGAAACAGGGTTAAGGGCTAGGACAGTCACAGGTGAGTCAGCCTCA CAAGAGCAACCTTCCCCTAGTGCAGA 5 GGAGGTCTCCTTTTGCCCCGGTTCCAACAAGAGAATGCAAGGCT Mouse;GTATCTCAATTTCCTTGAGCCTCTCTGTATTATAGAAGAAAAGT mm10: chr2:AGGGAAGCCATACGCCCCTTCTGAGCTTCAGTGTCTCTCTGTCT 36102524-CTGCAAATGAGGCTGGGGAGGCTGGGGGCGGGCGTGAAAGAG 36103193GCCCGCGCCAAGCCGACCCCCACCTCTGCCCCCTCCCCAGGTCAACAACCTCATCTGGCACGTGCGGTGCCTCGAGTGCTCCGTGTGTCGCACATCGCTGAGGCAGCAGAATAGCTGCTACATCAAGAACAAGGAGATCTACTGCAAGATGGACTACTTCAGGTAGGCAGCGGCCATCCCGCCAGCAAGCGCTGGAGCATGAACGCCTTGCACACGCGTGCCTAGGCCACTTGTGTGGCCTGTGCTCTCCAATTCCTGAGCCCTGCTGTTCAGAGTGCACAACGCGGCTCAGCGCACTGGCCCGGCCCTCCTACTCAGCACGTCTTACACAGAAGGGAGCGCCAGTCTCAGCCTGAGTTCTGGCGGGGGATCTGCCTCGGGTTCCTCCGATCTGACAGGCGCTGGCCACGGGTCTGGTTCCATCTCTGGTCTTTTCTGGCCCCGAGCACCAGTGTGTTCTGTTGAGCTCTGATGTCCGAG GCTCTGGCCCGGATCA  6CTCTGGCTACCTCTTATCTTGGGCATTCACGACAATTTCTAATTG Mouse;CAGGTAGTTTGTGTGTGTGCGCGTGTTTTTTTTCCCCCTCAGAGG mm10: chr2:CTTGGATTGCAAAGGAACTAAGCGATTACTTCAAGAGCCACGG 36103286-GTTAAGTGCAGGGAGAGGGGGAGAGAGAGGGAAAAAAACCCA 36104328ATCCAAATTCAAATTGCTTCATTAGAGAGACACCGCTTTTGTGGGGAAGGGCTTTAAATGCCCACTACAAAGTTAGGACTCATTGTTCAGCGCCGGTTTATATAACAGGCGAGGGGAGGCGCTGGGCTCTGACAGCTCCGAGCCAGTTCAGCAGCCGCCGTCGCCTGCATTCCCTCCCCCTCCCCCAGGTGATGGCCCAGCCAGGGTCCGGCTGCAAAGCGACCACCCGCTGTCTCGAAGGGACCGCTCCGCCTGCCATGGTGAGTCCTTTCGGTCCTGCTTTCGGCCCCGAGTCCCCCCAACAGCACAGGCCAGGGCTTCTGGCTCAGCCTTCCGGCTACCAACCTCTACCCCTGCGCTGGAAAACTGCCGATAGGAGCCGCCTCTCGTTGAGCCTTGGTTTTTCTGGCCTGGAATGTGAGCTTTGGCTGCTTCCTGCACCCAGGATGCGCTGTGTTAAAAGTTGGGGGCCGTCCCTTCTTCTCCAATAGGTCCTTTCATTCTTGTACTCCAGCCTAGGGCGCGACATCCCTGGCACATTTCGGTGTCAGTCGGTGCGCGAGGAAACCAGATTCAACTCTGAGTACTCGGCTAAGCGCTTCGCTGTTCCTCTCTCCCATTTCAGGCTCAGTCAGACGCAGAGGCCTTGGCAGGCGCTCTGGACAAGGACGAAGGTAGAGCCTCCCCATGTACGCCCAGCACACCGTCTGTCTGCTCGCCGCCCTCTGCTGCCTCTTCCGTGCCGTCTGCCGGCAAGAATATCTGCTCCAGTTGCGGTCTGGAGATCCTGGACCGGTATCTGCTCAAGGTGAGTCAGGGTAGGTGTGCCTGCTTGCCCACGGGTGTGGTTTGCAGCCCCAAGAGCTGT  7CAAGACTTTTAAAAGTTTAGATAAATAAACAAACATTTGACGGC Mouse;TTTCCATCACATCTAGACTATAATCCAAAGATCTATATGGTCCC mm10: chr2:AAACGACTTACACTTAACTACCGTCTCCCATATGGCTTCTTCCC 36114311-CCATCAGTCATTGTCCTCAGCCATAGTGGCCTCCCTGTTCCTTTG 36114817GGTACAAGGGAACAACTCCCTGAGAGGTTCCATTAGCTGCTGTTGCCTGAGATGCTCTTGAGCCCACACCATCTGCTCATTTCTCTCCTCACGTGTCAGTGATTAAGAGGCTGTCCTTGGCCTCCCGTCAAAATTACATCCCTGCCGCTTTCCACTTCTTGCCTTCTTATTTTCTAAATAGAACTAACTCACCACTACCCAACATTCTATATAATTGGATATCTGTCCTCTGTTTAAATATAATGTTGACTTCAAGAAAGAACGTTGTCACTGCCCTGTCACCAGACTTTTAAACAGTGCCTATCGTGTGG CACATGCTCAGTGAAATTG  8TCAACAGGGGGACACTTGGGAAAGAAGGATGGGGACAGAGCC Mouse;GAGAGGACTGTTACACATTAGAGAAACATCAGTGACTGTGCCA mm10: chr15:GCTTTGGGGTAGACTGCACAAAAGCCCTGAGGCAGCACAGGCA 78179109-GGATCCAGTCTGCTGGTCCCAGGAAGCTAACCGTCTCAGACAG 78179610AGCACAAAGCACCGAGACATGTGCCACAAGGCTTGTGTAGAGAGGTCAGAGGACAGCGTACAGGTCCCAGAGATCAAACTCAACCTCACCAGGCTTGGCAGCAAGCCTTTACCAACCCACCCCCACCCCACCCACCCTGCACGCGCCCCTCTCCCCTCCCCATGGTCTCCCATGGCTATCTCACTTGGCCCTAAAATGTTTAAGGATGACACTGGCTGCTGAGTGGAAATGAGACAGCAGAAGTCAACAGTAGATTTTAGGAAAGCCAGAGAAAAAGGCTTGTGCTGTTTTTAGAAAGCCAAGG GACAAGCTAAGATAGGGCCCAAGTAAT 9 AAATAGAACTGTGAGATAGGGGGAGAGGGGGCAGGAAGGACA Mouse;AGAGACCCCTGTCTCATTGTGATCCCCACCTGTCTGCTCTGTGG mm10: chr35:GAGGGTACCCATGAGGGCCAGCCCACAGCCCTTAGGTGGACAT 78195347-TGTCTGGTCCTGTCTCACTGTCCCTCCCAGCAGCCCCAGAGGCC 78196134AGGAGACAGGGGTCTCAGTCCTCACTGAGAGATGTGTAAACTGAGGCCCAGTGAATGTTGAGGGCCAGGGCATGCCCTTGGTGGGATGTGACCTGGGTCTCCTTCGCACGGGCTTCCTCCCCGAAGCCGAGCTGAGCATTTGGAGTTTGAAATGTTTCCGTACTTAGCAATCTGCTCCTCTATTCCCGGGCGGACTTCCGATAGCTCCGGCCTTATGCTGCACTAGATAAGATGGAGCAGGGAGAGGACACGGCACTACTTATGTAACCGGCCTCTTGAAAAATGGAGCAGCGGTCAGGGCGGAACAAGACGTCCTCTCTCTACGCATCCCTCTCCTTTCCCTGCTAAGGCTGCAGCTGGAGTCAGAGGCAGGGCTGTTCCAATCTGTCTTTGATCAGTAACGCAGCCAGCCTCCAGCCTCCGTCAGCCTCCTCATGGCTGAGACCCGGCCTCAGTTTCCCCCACTTACATCCCGAGGATCAGAGCCTGTGAGGATGAAATGGGATAAGGTAGCTGGAACCGTCTGGCAGAGAGCGAGTCCTCAGGACTGTTGATGCCTGTGGCTGCCTGGCTTGACCCCAAGTGACCCCGCCTCCTCATCCTGCAGCAGGA GAA 10TCTATAGAATGTGTCCCCAGCCTTGTTTTCCACACTTGATACGC Mouse;AAGGAATGCATACCACAGAGAGGGATGAGGGTAGCATCCAGCC mm10: chr15:TGCTTCCTGTGTGTCGGGGCGCTACAGCCACATCTCCCCAGTCC 78196305-ATCTCAGACCGTCACAGAGCTTCGCCGAATGTATAGCTTTGTTC 78196806TCTGTGCAGACAGGGAGACAGAGCCTTGGGAAGCATAGGTGCTTGCTTCTTTGCCCACTGAGTCTTAGCTGGACTTGCACACCACATGCCTCACAGCCGGGCGCACTTGCATTTGTCACCCAGGCCCAGTGATGATGGCTCTGCTTGCTTTGTGCTTTGTGCCAACTACAGCTCCAGCACCTGTGCCCTGGGTTTTCACTCCTTTAGTTGAACACGTAGTTACTGGGGTTGTAGGGATGGAGCCTTTCTGCTTCCTTCTGGCAAAGTCCTTAGCGGCCTGCTGCGGGGGTGGGGGGTGTTCAGGGGAG TGGTGATGAAGTATGACAG 11TCTCCAGTTGGAGAAACAGATGCTGTAACTGGGGCCACAGTAT Mouse;AAAGAGAGCCCAGACATTGAACTGTCAACACAGAAGCCTGGCA mm10: chr15:CACTGGAACTGGCAGTCCAGCTGGGAACAAGGGGTAGAGGCTG 78205234-AGGCCACTAAGTCAACTGAGGCAGGAGACATAGGAGCTAAAGC 78205766AGCTGAAGGGTGCAGGACAGCTGGGGGGTCTGAAGTGGGCCTCATGCCCAGAGCTATGAAGTCAGGGGCTGTAGCCTAGGAGCCTTGGAAGCCAGCTGGCAAGCTGTGGCCCAAAGACGCTGACTCACCAGGAGGGGGCAGCTGGAGCCAGGCACTCCTAAGGTTTCCAGGAAGGGCAGCCTTCCAGGGCTCAGCTAGGGGAGACAGTGTTGACAGCAAGTTGTCAGGCAACTTGAGCTACTGGGCAGCTGGGAAGCTGTCCCTTGGTCCCCAGTATCATCATCACCCCAGACGCTGCCCACCTGCCTCAGGTCCCACACAGTGATCCTCCCATCTTTAACACAAC ACATGACCAGAGAGA 12GTCACCCTCCCCCCAAACAACCCCTTCTTCTCTGGTTCGAGAAA Mouse;TTACAGGCATGAAAGATATAAATCGGGATGCTTGACTTGGGAA mm10: chr15:TATAAATCACTAAAGCTTGGGGGCAGGGGTGGGCGACCTTTGT 78224841-GACCGTCCTTGTGCGTGCCAGTAAATCCTGTGGTCCAGGGGAGA 78225364AGAAAAGGCTGTGTGGCTTCTGCTCACAAAGCTGCAGAAACCATTCTTTAAGCCCAAAAGCACTTCCAGAGAGAGCAGAGCATCCCCAGGCTGCTGGCTCAGCAAGTTCACTGTGCTCAATCTCAGGAAGTGAGGATAAGAGCAGTGCCTGGAGAGTGCCTGGTGCTGAGCTGAGGGTTTCTGAACACATTAAAGCGGGGAGCATGGACCGGGCCTCAGGAGGGGTGTTGAACATCCCTAGGCAGAGGAGTCTAGCTTCCTGGGAAAAGATATCAGGTTAAGCACACACATGTCCTCTGGAATAAGATAATCTTTCTGATCACACACTATACACACACAAAAGCCT GCTC 13GCCCTCTAGGCCACCTGACCAGGTCCCCTCAGTCCCCCCCTTCC Mouse;CACACTCCCACACTCAGCCCCCCTCCCCCCCCCCCGACCCCTGC mm10:chr15:AGGATTATCCTGTCTGTGTTCCTGACTCAGCCTGGGAGCCACCT 78241348-GGGCAGCAGGGGCCAAGGGTGTCCTAGAAGGGACCTGGAGTCC 78241856ACGCTGGGCCAAGCCTGCCCTTTCTCCCTCTGTCTTCCGTCCCTGCTTGCGGTTCTGCTGAATGTGGTTATTTCTCTGGCTCCTTTTACAGAGAATGCTGCTGCTAATTTTATGTGGAGCTCTGAGGCAGTGTAATTGGAAGCCAGACACCCTGTCAGCAGTGGGCTCCCGTCCTGAGCTGCCATGCTTCCTGCTCTCCTCCCGTCCCGGCTCCTCATTTCATGCAGCCACCTGTCCCAGGGAGAGAGGAGTCACCCAGGCCCCTCAGTCCGCCCCTTAAATAAGAAAGCCTCCGTTGCTCGGCACACA TACCAAGCAGCCGCTGGTGCAATCT14 GTGTTCTTCCCTTCCCCTTTGGACCCCCGAGACAAGCCAATAAA Mouse;ATACTCGGCAGGGTGGCTTCTCTCCTTTTTTTGCCAGTAATAAA mm10: chr9:CAGACTCAGAGCAAGTTAAGGGTCTGGTCCAAGGTCATGGCTG 107340928-GGATCAGTGACAGAGCCCAGAAGAGAACCTGAGACTTCTTGCT 107341325GAGCCAAGCTGGAGAGGACAGAAAGGAATGCGTCTACTCCATGCATGACCCTCTGCCAGCTTTGCTCCTTCCTAAGGGACCATGAACGATATGTGCACACCGCTCATACGTATGTGCACACCTGCAAGAGGAGGCATCCCATGTACACCTATGAGACGCACAGAGAAACATATATGTAGCCATAGGCTAGAAATTCTTTCTCTTTCTAGGTCTGCCCC TCTGCA 15GGACCACTCAGTGTACACGGAATGTAGAATTGAGTCTGCCATTG Mouse;GTCTTCCCTCAAAGTCTTGGAGGCTTGGGACTGATATTGGGAGC mm10: chr9:ATCTGGGCAGAGAAGGCCACAAAGACAGGGTGGTTTTTCTACA 107349227- CTGGGACATACTCGTGAGCATGCACAGAGGCGTGTCCCCAACTT 107350036CCCTGTCACCCCTGTCCTCTGCCGGCTAGAGGGGATGCGGGGGTGGACATATGCTGCTATTGGGCAGATATCACATGTTAAGAGGTGGGGGGGGGCTCAAGAGGCGGAGGGCTAGGAGCATCCCATGGGGAGAGGTTCTGGTTTTCTTGCTGCCTCTAGCTGCTATAAATACGTTAGCACTTGAGCAACTGGAAAGCTCTGAGTAATTTAGGATGCACAAAGCTGTAATTTAACTCCAGCATCTCAGTGTGCGAGAGCATTAAAGATGTAATTAAGATGTTTACACAAAGAGATTGGAGTCTGTGACACTTGGGGTGCAAAACCCCAGGAAGGGACACAATGGGTGAGGTGAGGATCTGTGGGAGGCCTGGGGACAGTCACTTGGATCCCAGCTATGAGATGGCAGGCCACCCAGCTGTTTCTCCTTGGAAATGTTTTGGCCTGGGGGTTGGGGGTGGGGCATCACACTTTGATATGGAGATGGGGCAACAAAGCCTGCAATATCTGGGGGTGGAGAGGTCAAGTGGATGGAGTCTTTTGAGATCATGTCAGGAAGAGGGCTCGATCCCCCAAAATCATGGTGACATATGGTGTCTCGGGGTTCACAGG AGCTATGTCTAAAATACAAAAGTAAA16 TCTGCAGAAGCCTGCCATTCCACCATTTAAACCTGTGACTCCAG Mouse;GCCTTAAGCCTGTTGAAGGTCGAGTCCCAGAAGGGTCATATGTG mm10: chr9:CAACTGCCTAGGGAGAGTTCCCACTCGCAGGGCCAAGAGGAGT 107399438-CCCCCGGTCTGAGGTGTGGGGGCGGGGACGTGCACTGGGCGCT 107399639GGGACCACGGCTGGGGCTCAGGACTCGC 17TGCCTCAGTTTCTTCGCCTAGAAAGCCGGGTCTAAGGGTACATG Mouse;CCCTGATTCTTTTCTGGGGTGTCTCGAATTTTAAACAACACATA mm10: chr9:CTGTTCTGGGCTGATGACAAGAGGAAGTACTGGTCGGTGGCTG 107443292-ATGGACATCCACCATGGTGGCAACTGGAGGGAGGGGGAACGGA 107444228CGTTGAAACCCTGCCCTCCTGGAATCTGTCGCATGCACGCACGTTGACAATGCTTGGCACTGGGGACAGGCTGGGATGGATGGAGCGGAGCGTGAGGAGGAGTGGGCATGCAGGCCCGAGTGTCTGTTTTGCTGATTGCTCCTTTTGCTTTCAAGGAGATTAAACTATTTTTAGTCCATGCCTACTGCTGGTGAGACGCTGGAGGAAGCCTTTCCATCGTTGAGATTTTCTGGAAGCTGCCAAGTGTGGTCTTCAGCTCAATTCTGGGAGCCTCCCAGAGTGGGAGGGAGGAACATTTCCATCTGGGGGCTTCGGGGACAGGCTAAGATCTTCCCTGGGGTCCTTGCTGCGCTGGCCTCCTCAAACCACGCTGCCTCGGCCTGCATAAAGCAGTAATCTGATGTGCCCGATGTTTGTAACGCTGTGTTTAAAAAAAGTAATTTATTTTCTAATTATTCCTTGTCTTGCATAACCATGCATTGCCAAAGTGTCGCTATTTAAAATATTTATCTCTCCACGCCGCAGGAGCAGCTCTGGAGCGTGGAGGGGGAAGAAATAAAAGTCCGCGTGCCAGTCGCAGGCATATTACTTTGACTCGTCCTGGTGGCTTTGACGTCTCCCTGTAAATACATTTATTTTTCATTAGGACGTTTCTGAGCTTGTGGCCCCCGGAGAGCGGAGTGATTACGCTGTTCATCTGCAAGCGATGCAATAGAGGGGTACTCGCAGAATGACTTCCGCCCAGA GCATCCTGCGCCTGTCT 18TAAAATACCTTATTTTTTTCCAGTCTCTAAACTGCTAATCTCCCA Mouse;GGCTAAGGGATTCTGGGACAAAGGCAAGGCCTGGAAGTGGAAA mm10: chr9:TCTGTAAAATTAGCTTCAGCGGTATTAGTGTTTGCAGTTGAAGA 107444825-TTGAAAAACTGCTTTCCCAGGGCCTGATTGGAGGCTCCACTCTC 107445746CTCCAGGAAGAGGCAAGGACTCTGGGCTGGCACTGAGGACAAATCCTGGGAGGCTGCTATGGGGCCTGGGAGCCAGGCTGCCTTGTGCTAGAGGCCTAGAGAGTGTCTGTGTCCCAAGTCCCAAGCTACCCCCAGCAGCTAACAGCTTTTCCAGTTCTCAGGCACAGCAGGTGCCAAGATCACGCTCTGGAGTCCAGCTGGGCCCCTTCCTCTTCTTTTTTTTTTTTTTTTTTTAAGACCTCCTGGACACTGTTCCTCTCCCCCCCCCCGTGACCCCCCCCCTCAGTTCTCAAACACGTGAGGGTTGGGGGAGGGTTCCACAGCCAGAGAGAGGGGCCAGCTCTGGTGCCTGTGGGTACGCCCGCCCGTATGGCCCATCAGGCCTCTTGTGTGCTTGATTGCCTCTGATTGGCTGCAGCTGAATTCAGCAAAAGCTATTATTTGCCCTTGATGAGCCAATCAGATGGCCTCATTGGCCATTCAGAGCAGGCACCGGAACCTGAGGGTGGGGTGGGGGGTGGGGGATGGAGATGGGACTCAGTGAGGGGGTGGGAAGCTCTAAAACAGATGCAGGACCTGAGCCTGTCTGTGTCCACCACGACCTTCACACAGGTCACACCCCCTTCCCCTGACTTGTCACCCCAAACCAGGGCTTGTTGCCCAACCCCACCTCACAATTCCCTCACTCTGTAACACCTTTCCATATACCTCTGCATGTCTAAACCCAAGACTTGCTCTATGAAATC 19AGACCCTGCTTAGCACAGCTCTTAGCGGGTCCTTTAGGGGGTCT Mouse;CCCAGCGGGCCCAGTGGGAATGAGATAAGGAAGGACACAGCTG mm10: chr9:TCCATTCTCCCGTGCCTGCTAAGGAGGAAATGGGGCCGCCTTAC 107452080-ATAATTGGGGCAATTTGTTCCACTCTTGTCCTCCTGGTATCATGG 107452718CTATCACCCCCTCCTTGCTCAGGGAGTCCTTGATTGAGCGAGAAGCTCAGGCCTCCCTCTCTCCCTCCTGCTGGGGGTTGCTGAACAGAGGGTGTAGGAGCCATAGGCTCTGTCACTGCTGAGATCTGCCAGATGTCTAGGCCAGGAGAAAATGGAAAGGGCTAAGTCACAGCATATGTGGCCACTCAGGCCTATAGCCCCAAATCTGCCTGGTAACCCATTATGTCCCCAGAGAATTTGCATGGGCGGACACCCTCATGCCGGGTCTCAGTAAGGGAAGGGGTGGGAGGCAAAAATATCCCTCCCCACCCTGAATCTCCACCCCCTCCCCCCAGAAACTGACACTTGGCCTTGTCTAAGGATGGGTTTTCCCAAAATCCTTCTGAAAAAAACAGAATTTCAAGAGTCACTCCCTCCGGGTCTCAGCCTAGAACATA TGCAGTATCCCCTGACGTCCATAGGG20 AAACTGGCACAGTAATGGCGGGCTGACAGACAAGGGAGTCTGT Mouse;AGCACCCGCTGCCTCCGCCCACCCCTTCTCCGAGCAATTAAAAG mm10: chr9:GTGTTTATGTGGGGCTGGCAGTGGCTTCTGCCTCCCTTCCATTAC 107470414-GAACATTAAGAGATCTTGACCCTTCCACTTTCCCCGCTCTTGAA 107471129AGGAGCTGCAGACACGTGGAGCCAATTAGGCGCACGCGTGGGCGCCAAGGGCCTGAGCAGCTTTTTCTCCCTGATTGCGGCGTTTACAGCTGATTATTCTCCCCTCACCCAAACAGTGCTGCTTCCTGGCAAGGTGCCACCCAGAGGAGCCGGCTGGGGGCCCCTGGGGACAGGGGAGGACTGGATTAGTAAATGGGCATCTATCGAATGGCTTTCATATGTGTGGCTGGAAGGGAGAAGGGTAGGGCCAGGAATGGTGGCAGCAAGGGCCCAGGTAGCAATGAGGGTTCTTCTAACCCACCATTTAGGGATAGCGATCAGAAAAGGGCCCTCGAGGAGGTGACCTAAATGTGTGTAGAAGCTGACGGCCACTACACACACACACACACACACACACACACATACACAAGCATCCTTGTCCTTGGAGTCGGTCAGCATGAGCAAGAGAAAGATGTTCCCAGTGGCCATGAGAGTGGAGCCCTCCTCCCTACTTACATCCAGGTTGGATGGCCAGGAGATCC TGAGATCCTTCAAGACTCC 21AAGCCACATCCTGGGTGGAAATATATGGCTTCAATTCCCACTCT Mouse;TCCGGATGACCTCTGTGGGGAGCCCTGGCTTCACCTTGGTCCAG mm10: chr9:CTTCATCCCTTAGCCTCGCTGCCAGGAAGGCAGTGAGGTCAGAG 107484887- GCTGGTGCTGGCGTG107485033 22 CCTACCTGGTGCCCGCCAACATCTGGGGGCCATCCTGGCCAGCG Mouse;CCAGCGTGGTGGTGAAGGCACTGTGCGCCGTGGTACTGTTTCTC mm10: chr9:TACCTGCTTTCCTTCGCTGTGGACACGGGCTGCCTGGCCGTCAC 107534490-CCCAGGCTACCTTTTCCCACCCAACTTCTGGATCTGGACCCTGG 107534786CCACCCACGGGCTCATGGAACAGCACGTGTGGGACGTGGCCATTAGCCTGGCCACAGTGGTTGTGGCCGGGCGATTACTGGAGCCCCTCTGGGGAGCCTTGGAGCTGCTCATCTTCTTCTC 23AAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGC Human;CTGGGGAGAGGGGGGCATAAATAACCCCTGCGTGCTGCACCAC hg19: AGGACCACGGTAAAT chr2:171672063- 171672163 24 GGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGHuman; CCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGG hg19: GGCCCAGGGCTGGAchr2: 171672697- 171672797 25GCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCG Human;GGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATA hg19: TATGGGAGGGGGAGG chr2:171672918- 171673018 26 TTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTCCTTCHuman; CGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAG hg19:CTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCC chr2:GCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCG 171673150-CCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGG 171673696AGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACC TCCCGAAATGAGTGCTTCCTGCCC 27CAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTC Human;CAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGC hg19: GCGCGCGGGGCTA chr2:171673900- 171674000 28 GAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAHuman; GAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCA hg19:CCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAG chr2:GGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCA 171674400-GACATGCCCGCCCCGTGCGCCCTCCCC 171674600 29GCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTC Human;AAGGGGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTG hg19:CAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTG chr2:CAGCCTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAG 171674903-AGGCCCCGGGACGACCGAGCTG 171675101 30AAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGC HumanGCATAAATAACCCCTGCGTGCTGCACCACCTGGGGAGAGGGGGAGGACCACGGTAAATGGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGAGCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATATGGGAGGGGGAGGTTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCCCAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCGCGCGCGGGGCTAGAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCACCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCCGCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGGGGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTGCAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTGCAGCCTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAGAGGCCCCGGGACGACCGAGCT G 31GGAGGAAGCCATCAACTAAACTACAATGACTGTAAGATACAAA HumanATTGGGAATGGTAACATATTTTGAAGTTCTGTTGACATAAAGAATCATGATATTAATGCCCATGGAAATGAAAGGGCGATCAACACTATGGTTTGAAAAGGGGGAAATTGTAGAGCACAGATGTGTTCGTGTGGCAGTGTGCTGTCTCTAGCAATACTCAGAGAAGAGAGAGAACAATGAAATTCTGATTGGCCCCAGTGTGAGCCCAGATGAGGTTCAGCTGCCAACTTTCTCTTTCACATCTTATGAAAGTCATTTAAGCACAACTAACTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTTGCCCAGGACAGAGTGCAGTAGTGACTCAATCTCGGCTCACTGCAGCCTCCACCTCCTAGGCTCAAACGGTCCTCCTGCATCAGCCTCCCAAGTAGCTGGAATTACAGGAGTGGCCCACCATGCCCAGCTAATTTTTGTATTTTTAATAGATACGGGGGTTTCACCATATCACCCAGGCTGGTCTCGAACTCCTGGCCTCAAGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTCAGCCACTATGCCCAACCCGACCAACCTTTTTTAAAATAAATATTTAAAAAATTGGTATTTCACATATATACTAGTATTTACATTTATCCACACAAAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGCGCATAAATAACCCCTGCGTGCTGCACCACCTGGGGAGAGGGGGAGGACCACGGTAAATGGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGAGCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATATGGGAGGGGGAGGTTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCCCAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCGCGCGCGGGGCTAGAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCACCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCCGCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGGGGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTGCAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTGCAGCCTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAGAGGCCCCGGGACGACCGAGCTG 32TCAACAGGGGGACACTTGGGAAAGAAGGATGGGGACAGAGCC Human andGAGAGGACTGTTACACATTAGAGAAACATCAGTGACTGTGCCA mouseGCTTTGGGGTAGACTGCACAAAAGCCCTGAGGCAGCACAGGCAGGATCCAGTCTGCTGGTCCCAGGAAGCTAACCGTCTCAGACAGAGCACAAAGCACCGAGACATGTGCCACAAGGCTTGTGTAGAGAGGTCAGAGGACAGCGTACAGGTCCCAGAGATCAAACTCAACCTCACCAGGCTTGGCAGCAAGCCTTTACCAACCCACCCCCACCCCACCCACCCTGCACGCGCCCCTCTCCCCTCCCCATGGTCTCCCATGGCTATCTCACTTGGCCCTAAAATGTTTAAGGATGACACTGGCTGCTGAGTGGAAATGAGACAGCAGAAGTCAACAGTAGATTTTAGGAAAGCCAGAGAAAAAGGCTTGTGCTGTTTTTAGAAAGCCAAGGGACAAGCTAAGATAGGGCCCAAGTAATGCTAGTATTTACATTTATCCACACAAAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGCGCATAAATAACCCCTGCGTGCTGCACCACCTGGGGAGAGGGGGAGGACCACGGTAAATGGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGAGCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATATGGGAGGGGGAGGTTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCCCAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCGCGCGCGGGGCTAGAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCACCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCCGCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGGGGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTGCAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTGCAGCCTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAGAGGCCCCGGGAC GACCGAGCTG 33ATTTACATTTATCCACACA Human 34 TGCCGCTGGACTCTCTTCCAAGGAACTAGGAGAACCAAGATCCMouse; GTTTTTCTGCCAAGGGCTGCCCCCCCCACGCCCCCAACCCCCTC chr9:ACCCCGATCCCCACAGAAAGAAATCTTGAGGTAGCTGGAGCTT 107,399,268-CTTCTGTGGGTGTGACAGGACTGCCATTCTCCTCTGTAGTCTGC 107,400,067AGAAGCCTGCCATTCCACCATTTAAACCTGTGACTCCAGGCCTTAAGCCTGTTGAAGGTCGAGTCCCAGAAGGGTCATATGTGCAACTGCCTAGGGAGAGTTCCCACTCGCAGGGCCAAGAGGAGTCCCCCGGTCTGAGGTGTGGGGGCGGGGACGTGCACTGGGCGCTGGGACCACGGCTGGGGCTCAGGACTCGCGAGCTTGGATTCGGATCGGTTTGCGCGAGCCAGTAGGGCAGGCTCCGGGGTGAACGGGGACGAGGGGCGCGCGGGCACAGGCGGGCGCGTGACCGCGGCGGGGGCGCGCGGAGGCGGGCCGGCCAAGGAGAGGGAGGGAGGGAATGAGGGAGGGAGCGACAGGGGAGGGCGGCGCCGGCAGGTTGGCGGCGGCCGCTATTTGAGCGCAGGTCCCGGGCCAGGCGCTCAAAGCGCTTGGAGCCAGCGCGGCGGGGAGATCGCTGCGCGCAGCCCGCAGAGGCGCTGCGGCCAGTGCAGCCCCGGAGGCCCCGCGCGGAGAAGGAGGTGGAGAAGAGGCCGGCTTTCCGCCCGCCGCCCGCGCCCCCCCACCTCCATCCCGCCGCCGCCGTCCCCCCTCCCTCCCC GCGGCGCCGCATCTTGAATGGAAAC35 GAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGT TTTTTTCTTCCATTTCAGGTGTCGTGA

In one aspect, regulatory elements disclosed herein are cell-typeselective. In some cases, regulatory elements disclosed herein areselective for PV neurons. In some cases, regulatory elements disclosedherein are selective for PV neurons in the CNS. In some cases, PV-cellselective regulatory elements or any regulatory elements disclosedherein can result in selective gene expression in PV neurons over atleast one, two, three, four, five, or more non-PV CNS cell-types.

In some cases, any one or more of the regulatory elements disclosedherein are operably linked to a transgene in an expression cassette toresult in selective expression in a target cell-type, e.g., a PV neuron.In some cases, a regulatory element of any of the embodiments hereincomprises or consists of any one of (i) SEQ ID NOs: 1-33; (ii) avariant, functional fragment, or a combination thereof; or (iii) asequence having at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to anyone of (i) or (ii). In some cases, a regulatory element comprises anyone of SEQ ID NOs: 1-32. In some cases, sequence identity is measured byBLAST.

In some cases, two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, or ten or moreof SEQ ID NOs: 1-32, or a functional fragment or a combination thereof,or sequences having at least 80%, at least 90%, at least 95%, or atleast 99% sequence identity thereto, are combined to form a largerregulatory element, or are operably linked to a gene in an expressioncassette. In some cases, two or more, three or more, four or more, fiveor more, six or more, seven or more, eight or more, nine or more, or tenor more of SEQ ID NOs: 1-32, or a functional fragment or a combinationthereof, or sequences having at least 80%, at least 90%, at least 95%,or at least 99% sequence identity thereto, are combined using a linkersequence of 1-50 nucleotides. In some cases, two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, or ten or more of SEQ ID NOs: 1-32, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 90%, at least 95%, or at least 99% sequence identity thereto, arecombined without a linker sequence. In some cases, a sequence of SEQ IDNO: 33 is used as a linker between any two regulatory elements. In somecases, a linker sequence between any two regulatory elements comprisesSEQ ID NO: 33 or a sequence having at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 33. In some cases, sequence identity ismeasured by BLAST.

In some cases, when two or more regulatory elements are combined or usedin an expression cassette, the regulatory elements need not be adjacentor linked in an expression cassette. For example, one regulatory elementcan be located upstream of a transgene, while a second regulatoryelement and/or additional regulatory elements can be located downstreamof the transgene. In some cases, one or more regulatory elements can belocated upstream of a transgene. In some cases, one or more regulatoryelements can be located downstream of a transgene.

In some cases, any one or more of SEQ ID NOs: 1-22, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 90%, at least 95%, or at least 99% sequence identity thereto, canbe combined with any one or more of SEQ ID NO: 23-30, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 90%, at least 95%, or at least 99% sequence identity thereto, toform a larger regulatory element. For example, a regulatory elementcomprises SEQ ID NO: 1 and SEQ ID NO: 30. A regulatory element comprisesSEQ ID NO: 8 and SEQ ID NO: 30. A regulatory element comprises SEQ IDNO: 1 and SEQ ID NOs: 23-29. A regulatory element comprises SEQ ID NO: 8and SEQ ID NOs: 23-29. In some cases, a regulatory element comprises SEQID NO: 30 or any one or more of SEQ ID NOs: 23-29, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 90%, at least 95%, or at least 99% sequence identity thereto.

In some cases, one or more regulatory elements of the present disclosureresult in selective gene expression in a PV cell. In some cases,regulatory elements that show selective activity or function in a targetcell type also show minimal activity or function in one or moreoff-target cell-types, e.g., non-PV CNS cell-types, non-inhibitoryneurons or excitatory neurons, non-PV cells.

In some cases, one or more regulatory elements operably linked to a genemodulates gene expression in a cell, including but not limited to,selective expression in a target cell-type over non-target cell-types.Selective expression in a target cell or cell type can also be referredto as cell-selective expression or cell-type selective expression.

Selective expression generally refers to expression in a high fractionof cells of the cell type of interest (or the target cell type) ascompared to other cells (or non-target cell type). Selective expressioncan also be viewed as preferential expression in a target cell or targetcell type over one or more non-target cells or cell-types. In somecases, selective expression of one or more regulatory elements of thisdisclosure is compared to CAG, EF1α, a constitutive promoter (e.g.,SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory elementthat is known to drive expression in any cell or cell type withoutselectivity. In some cases, selective expression of one or moreregulatory elements of this disclosure is compared to CAG, EF1α, aconstitutive promoter (e.g., SV40, CMV, UBC, PGK, and CBA), anon-selective regulatory element, or an expression cassette without theregulatory elements.

Non-target cell types can include a different subset, subtype, or typeof cells as compared to the target cell or target cell type, or allnon-target cell types. In some cases, one or more regulatory elementsoperably linked to a gene result in selective expression in a targetcell type over at least one type of non-target cells, or at least two,at least three, at least four, at least five, or more than five types ofnon-target cells. In some cases, non-target cell types refer to allother cell types not including the target cell type. In some cases,non-target cell types are all other cell types within a relevant tissueor organ not including the target cell type, e.g., all non-target celltypes in the CNS, all non-target cell types in the hippocampus. In somecases, a non-target cell or non-target cell type encompasses a subset orsubtype of cells that is not the target cell. For example, non-PV CNScell-types can include GABAergic cells that express calretinin and/orsomatostatin instead of parvalbumin, or all GABAergic cells that do notexpress parvalbumin. In some cases, cell types are distinguished byhaving a different cell marker, morphology, phenotype, genotype,function, and/or any other means for classifying cell types.

Selectivity of expression driven by a regulatory element in a cell orcell type of interest can be measured in a number of ways. Selectivityof gene expression in a target cell type over non-target cell types canbe measured by comparing the number of target cells that express adetectable level of a transcript from a gene that is operably linked toone or more regulatory elements to the total number of cells thatexpress the gene. Such measurement, detection, and quantification can bedone either in vivo or in vitro.

In some instances, selectivity for PV neurons can be determined using aco-localization assay. In some cases, the co-localization assay is basedon immunohistochemistry. In some cases, a detectable reporter gene isused as a transgene to allow the detection and/or measurement of geneexpression in a cell. In some cases, a detectable marker, e.g., afluorescent marker or an antibody, which specifically labels the targetcell is used to detect and/or measure the target cells. In some cases, aco-localization assay employs imaging, e.g., fluorescent imaging, todetermine the overlap between different fluorescent labels, e.g.,overlap between a fluorescence signal indicative of a target cell andanother fluorescence signal indicative of gene expression. In somecases, fluorescent labels used for a co-localization assay include a redfluorescent protein (RFP), such as a tdTomato reporter gene, and a greenfluorescent reporter protein, such as eGFP.

In some instances, a gene operably linked to one or more regulatoryelements is a fluorescent protein, e.g., eGFP or RFP, wherein expressionof the transgene provides a detectable signal. In some cases, tissue isstained for eGFP or fluorescence from eGFP is detected directly using afluorescence microscope. A second fluorescent marker or reporter genehaving a different fluorescence or detectable signal can be used toindicate the target cells, such as an antibody that identifies thetarget cells. For example, an anti-PV antibody that interactsspecifically with PV neurons can be used to yield a detectable signalthat is distinguishable from the fluorescence used to measure geneexpression, such as a red fluorescence or a red stain. Thus, in anexample wherein eGFP is a transgene operably linked to one or moreregulatory elements that drive selective expression in PV neurons, andwherein the PV neurons are labeled with an anti-PV antibody, selectivityof gene expression in PV cells is measured as percentage of eGFP+ cellsthat are also PV+. In such assay, PV+ cells that are also eGFP+ areindicated by the overlap of both fluorescence signals, i.e., an overlapof the red and green fluorescence. Such measurement, analysis, and/ordetection can be done by eye inspection or by a computer.

In some cases, one can also measure the proportion of a cell type ofinterest (or target cell type) that expresses a transgene as compared tothe proportion of non-target cell types (or other cells) that expressthe transgene to assess the selectivity of one or more regulatoryelements operably linked to the transgene. Similarly, selectivity ofexpression can also be measured by comparing the number of target cellsthat express a transgene operably linked to one or more regulatoryelements to the total number of all cells that express the transgene. Inboth approaches, the higher the number of target cells that express thetransgene, the more selective are the regulatory elements for the targetcells. In some cases, the target cells are PV neurons.

In some cases, one or more regulatory elements disclosed herein resultin increased selectivity in gene expression in a PV neuron. In somecases, one or more regulatory elements disclosed herein result inincreased selectivity in gene expression in PV neurons as compared tonon-PV CNS cell-types. In some cases, one or more regulatory elementsdisclosed herein result in increased selectivity in gene expression inPV neurons as compared to non-PV GABAergic cells, wherein non-PVGABAergic cells can be any one or more of GABAergic cells that expresscalretinin (CR), somatostatin (SOM), cholecystokinin (CCK), neuropeptideY (NPY), vasointestinal polypeptide (VIP), choline acetyltransferase(ChAT), or a combination thereof. In some cases, one or more regulatoryelements disclosed herein result in increased selectivity in geneexpression in PV neurons as compared to at least one, at least two, atleast three, at least four, at least five, or more than five non-PVGABAergic subtypes. In some cases, one or more regulatory elementsdisclosed herein result in increased selectivity in gene expression inPV neurons as compared to all other non-PV GABAergic cells, or all otherGABAergic cells that do not express PV, or all other CNS cells that donot express PV, or all other neurons that do not express PV. In somecases, one or more regulatory elements disclosed herein result inincreased selectivity in gene expression in PV neurons as compared toall non-PV cells in the CNS or all non-PV neurons.

In some cases, one or more regulatory elements operably linked to atransgene result in selective expression of the transgene in PV cells,wherein the percentage of PV cells expressing the transgene is at apercentage higher than gene expression in PV cells wherein the transgeneis operably linked to CAG, EF1α, a constitutive promoter (e.g., SV40,CMV, UBC, PGK, and CBA), or a non-selective regulatory element, such asSEQ ID NO: 34, or a functional fragment thereof, or a sequence having atleast 80% sequence identity thereto. In some cases, one or moreregulatory elements result in selective expression in PV neurons at alevel that is at least 1.5 fold, at least 2 hold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, atleast 20 fold, at least 25 fold, or at least 50 fold as compared toexpression of a gene operably linked to CAG, EF1a, a constitutivepromoter (e.g., SV40, CMV, UBC, PGK, and CBA), or a non-selectiveregulatory element such as SEQ ID NO: 34, or a functional fragmentthereof, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto.

In some aspects, a regulatory element is human derived or comprises asequence that is human derived. In some cases, a regulatory element ismouse derived or comprises a sequence that is mouse derived. In somecases, a regulatory element comprises a non-naturally occurringsequence. In some cases, a regulatory element is non-naturallyoccurring. In some cases, one or more human derived regulatory elementsare combined with another regulatory element to generate a non-naturallyoccurring regulatory element. In some cases, a human derived regulatoryelement is combined with a mouse derived regulatory element.

The term “human derived” as used herein refers to sequences that arefound in a human genome (or a human genome build), or sequenceshomologous thereto. A homologous sequence may be a sequence which has aregion with at least 80% sequence identity (e.g., as measured by BLAST)as compared to a region of the human genome. For example, a sequencethat is at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% homologous to a human sequence isdeemed human derived. In some cases, a regulatory element contains ahuman derived sequence and a non-human derived sequence such thatoverall the regulatory element has low sequence identity to the humangenome, while a part of the regulatory element has 100% sequenceidentity (or local sequence identity) to a sequence in the human genome.

In some cases, a human-derived regulatory element is a sequence that is100% identical to a human sequence. In some instances, the sequence of acell-type selective regulatory element is 100% human derived.

In other instances, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, or 99% of the regulatory element sequence is humanderived. For example, a regulatory element can have 50% of its sequencebe human derived, and the remaining 50% be non-human derived (e.g.,mouse derived or fully synthetic). For further example, a regulatoryelement that is regarded as 50% human derived and comprises 300 bp mayhave an overall 45% sequence identity to a sequence in the human genome,while base pairs 1-150 of the RE may have 90% identity (local sequenceidentity) to a similarly sized region of the human genome.

In some instances, a sequence that is homologous to a human derivedregulatory sequence is at least 90% identical to a human sequence. Insome cases, a regulatory element herein comprises a sequence that has atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to any one of SEQ ID NOs: 1, 23-31,and 33. In some cases, sequence identity is measured by BLAST. When aregulatory element comprises a sequence that is homologous (e.g., atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity) to any one or more of SEQ ID NOs: 23-29, or afunctional fragment or combination thereof, such regulatory elementresults in higher expression of an operably linked transgene (when apromoter is also present in the expression vector or cassette), ascompared to a similar vector without the regulatory element. Such higherexpression of a transgene can be observed, e.g., in HEK293T or CHOcells.

In some cases, one or more regulatory elements comprise any one or moreof SEQ ID NO: 23-29 combined with or used in combination with any one ormore of SEQ ID NOs: 1-22 with or without a linker sequence such as SEQID NO: 33. In some cases, any two regulatory elements of this disclosureare linked together using a polynucleotide linker comprising 1-50nucleotides, such as SEQ ID NO: 33 or a variant thereof. In some cases,the linker sequence is a human derived sequence. In some cases, thelinker sequence is mouse derived or non-naturally occurring. In somecases, two regulatory elements are joined without a linker or withoutany intervening sequence. In some cases, a linker comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides. In some cases, the linkersequence is a result of restriction enzyme site, ligation, PCR, and/orcloning.

In some cases, a human derived regulatory element is combined with amouse derived regulatory element, such as SEQ ID NO: 32, which is acombination of SEQ ID NO: 8 (a mouse derived sequence) and SEQ ID NOs:23-29 (human derived sequences). In some cases, cell-type selectiveregulatory elements are combined directly with no additional linkersequence. In other cases, cell-type selective regulatory elements arecombined with one or more short linker sequences which can be eitherdeliberate or cloning artifacts. In some cases, a linker sequencecomprises 1-50 bases. For example, SEQ ID NO: 31 comprises the sequenceof SEQ ID NOs: 1 and 23-29 along with an additional 19 bp of the genomicsequence (SEQ ID NO: 33) immediately following the sequence of SEQ IDNO: 1. SEQ ID NO: 32 also includes these 19 bp but without the sequenceof SEQ ID NO: 1. In other examples, the combined cell-type selectiveregulatory elements can include short sequences, generally less than 50bp, less than 20 bp, less than 15 bp, or less than 10 bp, from a cloningplasmid or restriction enzyme recognition site.

In some cases, regulatory elements can be derived from non-coding DNAsequences. In some cases, regulatory elements derived from non-codingDNA are associated with genes, such as upstream sequences, introns, 3′and 5′ untranslated regions (UTRs), and/or downstream regions. In othercases, regulatory elements derived from non-coding DNA sequences are notassociated with a gene. In some cases, regulatory elements are derivedfrom coding sequences. In some cases, the genomic region from which aregulatory element is derived is distinct from the genomic region fromwhich an operably linked transgene is derived. In some cases, a RE isderived from a distal genomic region or location with respect to thegenomic region or location from which the transgene is derived (such asa naturally occurring or an endogenous version of the transgene).

In one aspect, a regulatory element is any non-coding sequence thatmodulates gene expression, e.g., selectivity of expression in a targetcell. In some cases, the target cell is a PV neuron. In some cases, aregulatory element is derived from a genomic sequence upstream of atranscription initiation site, a 5′ UTR sequence, an exonic sequence, anintronic sequence, or a 3′ UTR sequence. In some cases, a human derivedregulatory element comprises an intronic human derived sequence. In somecases, a regulatory element comprises an enhancer, and its presence inan expression cassette along with a promoter increases expression of anoperably linked transgene in the target cell-type (e.g., PV neurons) ascompared to expression of the same transgene by the promoter without theenhancer. In some cases, an enhancer increases expression of an operablylinked transgene through either a transcriptional mechanism,posttranscriptional mechanism, or both. In some cases, a regulatoryelement comprises an enhancer sequence, a promoter sequence, or acombination of the enhancer and promoter sequences. In some cases, aregulatory element comprises one or more of a human derived enhancersequence, a human derived promoter sequence, a human derived intronicsequence, and/or a combination thereof.

In some cases, a regulatory element comprises one or more of SEQ ID NOs:1-32, or a fragment or a combination thereof, or sequences having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto. In some cases, a regulatory element comprisesone or more of SEQ ID NOs: 1-22, or a fragment or a combination thereof,or sequences having at least 80%, at least 85%, at least 90%, at least95%, or at least 99% sequence identity thereto. In some cases, aregulatory element comprises one or more of SEQ ID NOs: 23-29, or afragment or a combination thereof, or sequences having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto. In some cases, a regulatory element comprises one or more ofSEQ ID NOs: 30-32, or a fragment or a combination thereof, or sequenceshaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity thereto. In some cases, a regulatory elementcomprises a sequence of SEQ ID NOs: 30-32, or a fragment or acombination thereof, or sequences having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto.

In some instances, a regulatory element is derived from non-human DNAsequences or both human and non-human genomic sequences. In some cases,cell-type selective regulatory elements, or parts thereof, arehomologous to a mammalian genomic sequence. In some cases, theregulatory elements have at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to amammalian genomic sequence. In some cases, a regulatory element isderived from a mouse genomic sequence. In some cases, a regulatoryelement, or fragments thereof, has at least about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to amouse genomic sequence or a non-human mammalian genomic sequence. Insome cases, sequence identity is measured by BLAST. In some cases, theregulatory elements may comprise any of SEQ ID NOs: 1-33, or a fragmentor a combination thereof, or sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% sequence identitythereto.

In some cases, cell-type selective regulatory elements are short. Insome cases, the size of the regulatory elements is compatible with thecloning capacity of a vector, e.g., a viral vector or rAAV, such thatthe combined size of a transgene and one or more regulatory elementsdoes not exceed the cloning capacity of a vector. In some cases, thecell-type selective regulatory elements have a length of up to about2050 bp, 2000 bp, 1900 bp, 1800 bp, 1700 bp, 1600 bp, 1500 bp, 1400 bp,1300 bp, 1200 bp, 1100 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500bp, 400 bp, 300 bp, 200 bp, or 100 bp. In some cases, the cell-typeselective regulatory elements have a total length of no more than about20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp,300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1010bp, 1020 bp, 1030 bp, 1040 bp, 1050 bp, 1060 bp, 1070 bp, 1080 bp, 1090bp, 1100 bp, 1200 bp, 1300 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900bp, or 2000 bp. In some cases, the cell-type selective regulatoryelements have a length of about 100 bp-1100 bp, 100 bp-1000 bp, 100bp-900 bp, 200 bp-900 bp, 200 bp-800 bp, 300 bp-600 bp, 400 bp-800 bp,500 bp-600 bp, or 600 bp-900 bp. In some cases, a regulatory element isbetween about 400-600 bp, 400-600 bp, 400-700 bp, 400-800 bp, 400-900bp, 400-1000 bp, or 400-1500 bp. In some cases, a regulatory element isbetween about 500-600 bp, 500-700 bp, 500-800 bp, 500-900 bp, 500-1000bp, or 500-1500 bp. In some cases, two or more regulatory elements arecombined to form a larger cell-type selective regulatory element of1300-2500 bp, 1300-2060 bp, about 1350 bp, about 2050 bp, or about 1880bp.

In some cases, two or more cell-type selective regulatory elements canbe combined. For example, two, three, four, five, six, seven, eight,nine, ten or more cell-type selective regulatory elements can becombined. For example, SEQ ID NO: 30 comprises sequences from sevenregulatory elements, i.e., SEQ ID NOs: 23-29, all of which are derivedfrom human genomic sequence. In some cases, cell-type selectiveregulatory elements refer to PV-neuron selective regulatory elements.

In some cases, a cell-type selective regulatory element is repeated twoor more times to make a combined regulatory element that is alsocell-type selective or has enhance cell-type selective property. In somecases, two or more regulatory elements with different cell-typeselectivity are combined. In some cases, a cell-type selectiveregulatory element is combined with a non-selective regulatory element,e.g., a non-selective enhancer element that drives high gene expression.For example, a promoter regulatory element with high selectivity for atarget cell can be combined with a regulatory element with highefficiency of expression. In some cases, one or more cell type-selectiveregulatory elements are combined with one or more high efficiencyregulatory elements. For example, any one or more of SEQ ID NOs: 1-32,or a fragment or a combination thereof, or sequences having at least80%, at least 85%, at least 90%, at least 95%, or at least 99% sequenceidentity thereto, can be combined with a constitutive promoter, such asa GAD2 promoter, a human synapsin promoter, a minCMV promoter, a TATAbox, a super core promoter, or an EF1α promoter, or a combinationthereof.

In some aspects, the present disclosure provides a list of regulatoryelements that can be added to any gene therapy to result in selectivegene expression in a target cell type, such as a PV neuron over one ormore non-target cell types, such as non-PV CNS cells, including but notlimited to excitatory cells and/or non-PV GABAergic cells.

In some cases, cell-type selective regulatory elements can be combinedwith other regulatory elements such as a high expressing promoter or asequence that increases mRNA stability. In some cases, one or morecell-type selective regulatory elements are combined with a human, anon-human, or a non-mammalian sequence, for example a hSyn1 promoter,CBA promoter, a CMV promoter, an EF1α promoter, a polyA signal (e.g.,SV40 polyA signal), or a post-transcriptional regulatory element such aswoodchuck hepatitis virus post-transcriptional regulatory element(WPRE).

In some cases, the combined regulatory elements can come from differentspecies. The combined regulatory elements can come from differentgenomic regions within a species. In some cases, regulatory elements arederived from distal genomic sequences, e.g., sequences that do notnormally or naturally associate with each other or with a cell type ofinterest, are combined. In some cases, individual regulatory elementsused to make a combined regulatory element can come from different humanchromosomes.

In one aspect, a regulatory element of the disclosure comprises afunctional fragment of any of SEQ ID NOs: 1-32, or a combinationthereof, or sequences having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto. Such functionalfragment can increase expression of a transgene in an expressioncassette or vector when compared to a similar expression cassette orvector without the regulatory element. Such a functional fragment canfunction as an enhancer to increase cell-type selective expression whenthe fragment is operably linked to a transgene as compared to a similarvector or cassette without the functional fragment. A fragment ispreferably more than 30, 40, 50, or 60 bp in length.

In some cases, a PV cell selective regulatory element or any regulatoryelement of this disclosure comprises any one of SEQ ID NOs: 1-32, (ii) anucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to any one of SEQ ID NOs: 1-32, (iii) a functionalfragment of any sequence of (i) or (ii), or (iv) a combination of anysequence of (i), (ii) and/or (iii). In some cases, sequence identity ismeasured by BLAST. In some cases, two or more of SEQ ID NOs: 1-29, or afunctional fragment or a combination thereof, or sequences having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto, are used as a regulatory element to increasetransgene expression selectively in PV cells as compared to non-PV CNScells, or to in any target cell type as compared to non-target celltype. In some cases, a functional fragment is one that results inselective expression in a target cell type over one or more non-targetcell types.

In some cases, two or more copies of a regulatory element can be used toenhance selective expression in a target cell, e.g., two or more copiesof any one of SEQ ID NOs: 1-29, or a fragment or a combination thereof,or sequences having at least 80%, at least 85%, at least 90%, at least95%, or at least 99% sequence identity thereto.

In other cases, one or more of SEQ ID NOs: 1-32, or a fragment or acombination thereof, or sequences having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto, areoperably linked to another regulatory element, such as a promoter orenhancer, to further increase selective expression in a target cell. Insome cases, one can enhance any gene therapy by adding one or moreregulatory elements as disclosed herein to improve or increaseexpression from the gene therapy in a target cell as compared tonon-target cells. In some cases, the target cell is PV neurons orGABAergic cells that express parvalbumin.

In some aspects, one or more regulatory elements (e.g., any one or moreof SEQ ID NOs: 1-32, or a fragment or a combination thereof, orsequences having at least 80%, at least 85%, at least 90%, at least 95%,or at least 99% sequence identity thereto) disclosed herein showcell-type selectivity for a target cell type over at least one, at leasttwo, at least three, at least four, at least five, or more than fivenon-target cell types. In some cases, a regulatory element drivesselective expression or preferential expression in a target cell subtypeover at least one, at least two, at least three, at least four, at leastfive, or more than five non-target subtypes, or all other known subtypesof the cell. For example, GABAergic cells comprise different subtypes,including PV cells. In some cases, the target cell type is a PV cell. Insome cases, one or more regulatory elements are selective for PV cellsover at least one, at least two, at least three, at least four, at leastfive, or more than five non-target cell types. In some instances, one ormore regulatory elements are selective for PV cells over all other knownCNS cell-types.

In some cases, any one or more of SEQ ID NOs: 1-32, or a fragment or acombination thereof, or sequences having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto, canbe combined with any one or more of SEQ ID NOs: 1-32, or a fragment or acombination thereof, or sequences having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto. Insome cases, such combined regulatory elements are linked using a linkerof 1-50 nucleotides. In some cases, such combined regulatory elementsare not linked.

In some cases, one or more regulatory elements disclosed herein, whenoperably linked to any transgene (e.g., a reporter transgene or atherapeutic transgene), drives selective expression or preferentialexpression in at least one target cell type at a level that isstatistically significantly higher than the expression driven by CAG,EF1α, a constitutive promoter (e.g., SV40, CMV, UBC, PGK, and CBA), or anon-selective regulatory element (e.g., SEQ ID NO: 34, or a fragmentthereof, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto) when operablylinked to the same transgene, or by the same construct without theregulatory elements. In some cases, statistically significantly highermeans the regulatory elements drive selective expression in the targetcell type at a level that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 times the expression level by CAG, EF1α,a constitutive promoter (e.g., SV40, CMV, UBC, PGK, and CBA), or anon-selective regulatory element when operably linked to the sametransgene, or by the same construct without the regulatory elements. Insome cases, such cell-type selective expression is assayed using aco-localization assay as described herein. In some cases, the targetcell type is a parvalbumin cell. In some cases, such co-localizationassay is conducted using an anti-PV antibody. In some cases, suchco-localization assay is conducted using a PV-Cre mouse as disclosedherein. In some cases, the non-selective regulatory element is SEQ IDNO: 34, or a fragment thereof, or a sequence having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto.

Expression Cassettes

The terms “expression cassette” and “nucleic acid cassette” are usedinterchangeably to refer to a polynucleotide molecule or a nucleic acidsequence. In some cases, an expression cassette comprises one or moreregulatory elements disclosed herein operably linked to a transgene. Insome cases, an expression cassette comprises one or more regulatoryelements. In some cases, an expression cassette comprises one or morecell type selective regulatory elements disclosed herein. In some cases,an expression cassette comprises one or more PV cell selectiveregulatory elements disclosed herein. In some cases, the expressioncassette further comprises a promoter. In some cases, an expressioncassette comprises one or more sequences of SEQ ID NOs: 1-32 and/or anycombination thereof. In some cases, an expression cassette comprises oneor more of SEQ ID NOs: 1-32, (ii) a nucleic acid sequence having atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQID NOs: 1-32, (iii) a functional fragment of any sequence of (i) or(ii), or (iv) a combination of any sequence of (i), (ii) and/or (iii).In some cases, sequence identity is measured by BLAST. In some cases, aregulatory element is located upstream of a transgene in an expressioncassette. In some cases, a regulatory element is located downstream of atransgene in an expression cassette. In some cases, an expressioncassette further comprises a promoter, e.g., a hSyn1 promoter, CBApromoter, a CMV promoter, an EF1α promoter, a polyA signal (e.g., SV40polyA signal), or a post-transcriptional regulatory element such aswoodchuck hepatitis virus post-transcriptional regulatory element(WPRE).

In some aspects, one or more regulatory elements described herein areoperably linked to a transgene in an expression cassette. In some cases,a gene therapy comprises an expression cassette comprising a transgeneoperably linked to one or more, two or more, three or more, four ormore, or five or more regulatory elements of the present disclosure toresult in selective expression of the transgene in a target tissue orcell type, such as PV neurons. In some cases, an expression cassettecomprises one or more PV cell selective regulatory elements or one ormore regulatory elements disclosed herein operably linked to atransgene, e.g., a reporter gene, eGFP, SCN1A, SNC2A, SNC8A, SCN1B,SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, a DNA binding protein, or a variantor a fragment thereof, or sequences having at least 80%, at least 85%,at least 90%, at least 95%, or at least 99% sequence identity thereto.

In some cases, an expression cassette is adapted for delivery via genetherapy. In some cases, an expression cassette is a linear or a circularconstruct. In some cases, an expression cassette is part of a plasmid,vector, a viral vector, or rAAV.

In some cases, a gene therapy is administered directly to the CNS of asubject in need thereof or systematically via injection and/or infusion.In some cases, such subject has been diagnosed with a disease orcondition associated with a haploinsufficiency or a genetic mutation,such as a haploinsufficiency or a mutation in any one of the followinggenes: SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, orSTXBP1. In some cases, the subject is at risk for or has Dravetsyndrome, Alzheimer's disease, epilepsy, neurodegeneration, tauopathy,neuronal hypoexcitability and/or seizures. In some cases, such genetherapy is delivered using a virus or a viral vector, such as rAAV. Insome cases, an AAV serotype with a tropism for CNS cells and/or abilityto cross the blood brain barrier is used, such as AAV9 or a variantthereof.

In some cases, one or more regulatory elements (e.g., one or more of SEQID NOs: 1-32, or a fragment or a combination thereof, or sequenceshaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity thereto) operably linked to a transgene inan expression cassette result in selective gene expression in PV cellsas compared to non-PV CNS cells, or as compared to a control element,such as a CAG, EF1α, a constitutive promoter (e.g., SV40, CMV, UBC, PGK,and CBA), or a non-selective regulatory element (e.g., SEQ ID NO: 34, ora functional fragment thereof, or a sequence having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto). In some cases, regulatory elements result in at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% of all cellsexpressing the transgene are PV neurons. In some cases, regulatoryelements result in selective gene expression in PV neurons that is about1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 7.5times, 8 times, 9 times, or 10 times higher than expected for naturaldistribution of PV neurons in CNS. In some cases, a regulatory elementdrives selective expression in PV cells, wherein the percentage of PVcells expressing the transgene is at a percentage that is at least 1.5fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9fold, or at least 10 fold higher than the expected distribution of PVcells in the CNS, or at least 1-5%, 5%-10%, 10-15%, 15-20%, 20-25%,25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 65-70%, 70-75%,75-80%, 80-85%, 85-90%, or 90-95% higher than the expression in PV cellswhen the transgene is operably linked to CAG, EF1α, a constitutivepromoter (e.g., SV40, CMV, UBC, PGK, and CBA), or a non-selectiveregulatory element having a sequence of SEQ ID NO: 34, or a functionalfragment thereof, or a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto, andas measured in an immunohistochemical co-localization assay. In somecases, a regulatory element in an expression cassette, or use of suchregulatory element in an expression cassette, results in selective geneexpression in PV cells, or PV cells in the CNS, or PV neurons, whereinabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95% of the cells expressing the transgene are PVpositive.

In some cases, an expression cassette or a gene therapy comprises one ormore, two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, or ten or moreregulatory elements as described in TABLE 1, e.g., SEQ ID NOs: 1-32, ora functional fragment or a combination thereof, or sequences having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto.

In some cases, one or more PV cell selective regulatory elements or oneor more regulatory elements disclosed herein are operably linked to anytransgene in an expression cassette. In some cases, the expressioncassette is a gene therapy. In some cases, the expression cassette ispart of a vector or a plasmid, e.g., a viral vector or rAAV vector. Insome cases, the expression cassette is part of AAV1, AAV8, AAV9, orAAVDJ or a variant or hybrid thereof. In some cases, the expressioncassette comprises one or more PV cell selective regulatory elements orone or more regulatory elements disclosed herein operably linked to atransgene, wherein the transgene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B,KV3.1, KAV3.2, KV3.3, STXBP1, DNA binding protein (e.g., transcriptionalmodulator of an endogenous gene), or a variant or a functional fragmentthereof, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto. In some cases,such regulatory elements increase the selective expression of thetransgene in PV neurons as compared to non-PV CNS cell-types. In somecases, such regulatory elements increase the expression of the transgeneselectively in a target cell type, such as a PV neuron. In some cases,the target cell type is a PV cell.

Techniques contemplated herein for gene therapy of somatic cells includedelivery via a viral vector (e.g., retroviral, adenoviral, AAV,helper-dependent adenoviral systems, hybrid adenoviral systems, herpessimplex, pox virus, lentivirus, and Epstein-Barr virus), and non-viralsystems, such as physical systems (naked DNA, DNA bombardment,electroporation, hydrodynamic, ultrasound, and magnetofection), andchemical system (cationic lipids, different cationic polymers, and lipidpolymers).

The cloning capacity of vectors or viral expression vectors is aparticular challenge for expression of large transgenes. For example,AAV vectors typically have a packaging capacity of ˜4.8 kb, lentivirusestypically have a capacity of ˜8 kb, adenoviruses typically have acapacity of ˜7.5 kb and alphaviruses typically have a capacity of ˜7.5kb. Some viruses can have larger packaging capacities, for exampleherpesvirus can have a capacity of >30 kb and vaccinia a capacity of ˜25kb. Advantages of using AAV for gene therapy include low pathogenicity,very low frequency of integration into the host genome, and the abilityto infect dividing and non-dividing cells.

To address the size constraints of certain viral vectors or to improveexpression from viral vectors, the present disclosure contemplates theuse of regulatory elements that are shorter than 2.5 kb, 2 kb, 1.5 kb, 1kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 150bp, or 110 bp, but at least 10 bp, 50 bp or 100 bp in length. In somecases, the size of the combined regulatory element is about 2500 bp,2000 bp, 1500 bp, 1400 bp, 1300 bp, 1200 bp, 1100 bp, or 1000 bp. Insome cases, each combined regulatory element has a total length of about100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp,1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp,1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, or 2500bp. In some cases, the size of a combined RE has a total length of about200 bp-3000 bp, 200 bp-2500 bp, 200 bp-2100 bp, 500 bp-2500 bp, 1000bp-2500 bp, 1500 bp-2500 bp, 1500b-2000 bp, or 2000 bp-2500 bp.

In some cases, a regulatory element of the disclosure is preferably (i)one that selectively drives expression in a cell-type of interest, suchas PV cells; (ii) includes a human derived sequence, and (iii) issmaller than 2.5 kb, 2 kb, 1.5 kb, or 1 kb.

Also contemplated herein are expression cassettes, which can be acircular or linear nucleic acid molecule. In some cases, an expressioncassette is delivered to cells (e.g., a plurality of different cells orcell types including target cells or cell types and/or non-target celltypes) in a vector (e.g., an expression vector). A vector can be anintegrating or non-integrating vector, referring to the ability of thevector to integrate the expression cassette and/or transgene into agenome of a cell. Either an integrating vector or a non-integratingvector can be used to deliver an expression cassette containing atransgene operably linked to a regulatory element. Examples of vectorsinclude, but are not limited to, (a) non-viral vectors such as nucleicacid vectors including linear oligonucleotides and circular plasmids;artificial chromosomes such as human artificial chromosomes (HACs),yeast artificial chromosomes (YACs), and bacterial artificialchromosomes (BACs or PACs); episomal vectors; transposons (e.g.,PiggyBac); and (b) viral vectors such as retroviral vectors, lentiviralvectors, adenoviral vectors, and AAV vectors. Viruses have severaladvantages for delivery of nucleic acids, including high infectivityand/or tropism for certain target cells or tissues. In some cases, avirus is used to deliver a nucleic acid molecule or expression cassettecomprising one or more regulatory elements, as described herein,operably linked to a transgene.

Preferred characteristics of viral gene therapy vectors or gene deliveryvectors include the ability to be reproducible and stably propagated andpurified to high titers; to mediate targeted delivery (e.g., to deliverthe transgene specifically to a tissue or organ of interest withoutwidespread vector dissemination elsewhere or off-target delivery); andto mediate gene delivery and/or transgene expression without inducingharmful side effects or off-target effects. To avoid potential harmfulside effects, targeted expression or tissue/cell type selectiveexpression can be achieved by placing the transgene under the control ofa cell-type-selective regulatory element, e.g., one or more of SEQ IDNOs: 1-32, or a functional fragment or a combination thereof, orsequences having at least 80%, at least 85%, at least 90%, at least 95%,or at least 99% sequence identity thereto, an enhancer, promoter,stability element, UTR, or a combination thereof. For example, viralparticles containing a viral vector can be designed to infect manydifferent cell types but expression of the transgene is enhanced and/oroptimized in a cell type of interest (e.g. PV neurons), and expressionof the transgene is reduced and/or minimized in other non-target celltypes (e.g., non-PV CNS cells). The differential expression of thetransgene in different cell types can be controlled, engineered, ormanipulated using different transcription factors or regulatory elementsthat are selective for one or more cell types. In some cases, one ormore regulatory elements, such as a promoter or enhancer, or acombination thereof, are operably linked to a transgene to drive tissue-or cell-selective expression of the transgene. In some cases, one ormore regulatory elements used in a gene therapy or a vector drive geneexpression in a cell type selective manner, i.e., confer selective geneexpression in a target cell, cell type, or tissue, and/or do not drivegene expression in one or more (e.g., at least one, two, three, or four)off-target cells or cell types. In some cases, one or more regulatoryelements operably linked to a transgene enhances selective expression ofthe transgene in a target cell, cell type, or tissue, while the one ormore regulatory elements suppress transgene expression in off-targetcells, cell type, or tissue, or confers significantly lower, de minimis,or statistically lower gene expression in one or more off-target cells,cell types, or tissue.

Several serotypes of AAV, non-pathogenic parvovirus, have beenengineered for the purposes of gene delivery, some of which are known tohave tropism for certain tissues or cell types. Viruses used for variousgene-therapy applications can be engineered to be replication-deficientor to have low toxicity and low pathogenicity in a subject or a host.Such virus-based vectors can be obtained by deleting all, or some, ofthe coding regions from the viral genome, and leaving intact thosesequences (e.g., inverted terminal repeat sequences) that are necessaryfor functions such as packaging the vector genome into the virus capsidor the integration of vector nucleic acid (e.g., DNA) into the hostchromatin. An expression cassette comprising a transgene, for example,can be cloned into a viral backbone such as a modified or engineeredviral backbone lacking viral genes, and used in conjunction withadditional vectors (e.g., packaging vectors), which can, for example,when co-transfected, produce recombinant viral vector particles. In somecases, an AAV serotype that can cross the blood brain barrier or infectcells of the CNS is preferred. In some cases, AAV9 or a variant thereofis used to deliver an expression cassette of this disclosure, comprisingone or more PV selective regulatory elements operably linked to atransgene.

One advantage of delivering expression cassettes of this disclosureusing gene therapy, e.g., rAAV, as described herein, is that suchtherapies can provide more targeted and sustained therapeutic effectsover time. Additionally, viral gene therapies can be engineered to havetropism for a cell type or tissue of interest over non-target cell typesor tissues. For example, viral gene therapies can be engineered toinfect and deliver a payload or a therapeutic agent, e.g., atranscriptional modulator or a transgene, to one or more regions,tissues, or cell types within the CNS (e.g., PV cells), while havingminimal effects on off-target tissues or cell types (e.g., non-CNStissue or cell types, non-PV CNS cells). In some cases, viral genetherapies can be engineered to deliver a transgene across the bloodbrain barrier and/or target a specific region or tissue within the CNS(e.g., hippocampus) or a cell type within the CNS, e.g., PV cells.

In some cases, an AAV vector or an AAV viral particle, or virion, usedto deliver one or more regulatory elements and a transgene into a cell,cell type, or tissue, in vivo or in vitro, is preferablyreplication-deficient. In some cases, an AAV virus is engineered orgenetically modified so that it can replicate and generate virions onlyin the presence of helper factors.

In some cases, the expression cassette is designed for delivery by anAAV or a recombinant AAV (rAAV). In some cases, an expression cassetteis delivered using a lentivirus or a lentiviral vector. In some cases,larger transgenes, i.e., genes that exceed the cloning capacity of AAV,are preferably delivered using a lentivirus or a lentiviral vector.

The AAV used in the compositions and methods described herein can be ofany serotype (e.g., AAV1, AAV2, AAV5, AAV8, AAV9, and AAVDJ), includinghybrid or chimeric AAV serotypes. In some cases, AAV is used to deliverand/or express a transgene operably linked to one or more regulatoryelements that are selective for PV neurons as compared to non-PV CNScells. In some cases, an AAV with a high tropism for CNS cells and/orcrosses the blood brain barrier is used. In some cases, AAV1, AAV8,AAV9, and/or AAVDJ are used to deliver to deliver an expression cassettedescribed herein.

In some cases, an expression cassette comprises one or more PV cellselective regulatory elements or one or more regulatory elementsdisclosed herein operably linked to a transgene that is known to beinsufficiently expressed in vivo, such as in a disease or conditionassociated with haploinsufficiency in the gene. In some cases, thetransgene is a voltage-gated ion channel (e.g., a sodium ion channel ora potassium ion channel), a neurotransmitter regulator, or a subunit orfunctional fragment thereof. In some aspects, the transgene is a DNAbinding protein, an ion channel, a neurotransmitter regulator, or asubunit of the ion channel or neurotransmitter regulator. In some cases,the transgene is a DNA binding protein that comprises one or more zincfingers. In some cases, the DNA binding protein comprises a domain ofCas9, a Cas family protein, nuclease-inactivated Cas9 (or dCas9), a dCasfamily protein, or a transcriptional activator like effector (TALE). Insome cases, the transgene is a DNA binding protein that comprises a DNAbinding domain of a DNA binding protein or a DNA cleaving protein (e.g.,a nuclease, a restriction enzyme, a recombinase, etc.) wherein the DNAcleaving domain or nuclease domain has been deactivated, e.g., anuclease-deactivated Cas (dCas), a deactivated transcriptionactivator-like effector nuclease, or a nuclease-deactivated zinc fingerprotein. In some cases, the DNA binding domain is linked to atranscriptional modulating domain (e.g., a transcriptional activator orrepressor domain). In some cases, the transgene comprises a gene editingprotein, e.g., a Cas protein, Cas9.

In some aspects, the transgene is a voltage-gated ion channel or asubunit thereof, such as SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1,KV3.2, or KV3.3, or a functional fragment or variant thereof. In somecases, the transgene is an alpha subunit of a sodium ion channel. Insome cases, the transgene is a beta subunit of a sodium ion channel. Insome aspects, the neurotransmitter regulator is STXBP1 or a functionalfragment or variant thereof.

In some aspects, an expression cassette is delivered as a viral vector,such as AAV. In some aspects, the AAV is AAV1, AAV8, AAV9, AAV-DJ,scAAV1, scAAV8, or scAAV9. In some aspects, a gene therapy comprising anexpression cassette of this disclosure is administered to a subject inneed thereof (e.g., a human patient, a mammal, a transgenic animal, oran animal model). In some cases, the subject in need thereof hassymptoms of, has been diagnosed with, or is at risk of developingAlzheimer's disease, Dravet syndrome, epilepsy, neurodegeneration,tauopathy, neuronal hypoexcitability, and/or seizures. In some cases,the subject in need thereof has an insufficient gene expression or amutation in any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1,KV3.2, KV3.3, and STXBP1.

In some cases, a gene therapy, such as rAAV9, is used to deliver anexpression cassette comprising one or more PV cell selective regulatoryelements or one or more regulatory elements disclosed herein operablylinked a transgene, wherein the transgene is SCN1A, SNC2A, SNC8A, SCN1B,SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, a DNA binding protein, or afunctional fragment thereof, or a sequence having at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to any one of SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, a DNA binding protein,or a functional fragment thereof. In some cases, the transgene comprisesa sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8′7%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 37-43, or a functional fragmentthereof, as provided in TABLE 2 below.

In some cases, the transgene is any one of SEQ ID NOs: 36-43, or afunctional fragment thereof, or sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% sequence identitythereto. In some cases, one or more regulatory elements disclosed hereinare operably linked to any one of SEQ ID NOs: 36-43 in an expressioncassette, or a functional fragment thereof, or sequences having at least80%, at least 85%, at least 90%, at least 95%, or at least 99% sequenceidentity thereto.

TABLE 2 List of amino acid sequences disclosed herein. SEQ ID NO. GeneAmino Acid Sequence 36 eGFPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL 37 SCN1BMGTLLALVVGAALVSSAWGGCVEVDSDTEAVYGMTFKILCISCKRRSETTAETFTEWTFRQKGTEEFVKILRYENEVLQLEEDERFEGRVVWNGSRGTKDLQDLSIFITNVTYNHSGDYECHVYRLLFFDNYEHNTSVVKKIHLEVVDKANRDMASIVSEIMMYVLIVVLTIWLVAEMVYCYKKIAAATEAAAQENASEYLAITSESKENCTGVQVAE 38 SCN2BMHRDAWLPRPAFSLTGLSLFFSLVPPGRSMEVTVPATLNVLNGSDARLPCTFNSCYTVNHKQFSLNWTYQECNNCSEEMFLQFRMKIINLKLERFQDRVEFSGNPSKYDVSVMLRNVQPEDEGIYNCYIMNPPDRHRGHGKIHLQVLMEEPPERDSTVAVIVGASVGGFLAVVILVLMVVKCVRRKKEQKLSTDDLKTEEEGKTDGEGNPDDGAK 39 SCN1AMEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMIEQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEVIIDKPATDDNGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFWAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK 40 STXBP1MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMTDIMTEGITIVEDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTDSCPDALFNELVKSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKNPILERLAEQIATLCATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGPDKARSQLLILDRGFDPSSPVLHELTFQAMSYDLLPIENDVYKYETSGIGEARVKEVLLDEDDDLWIALRHKHIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKKMPQYQKELSKYSTHLHLAEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPILLDANVSTYDKIRIILLYIFLKNGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDSTLRRRSKPERKERISEQTYQLSRWTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTAVSARYGHWHKNKAPGEYRSGPRLIIFILGGVSLNEMRCAYEVTQANGKWEVLIGSTHILTPTKFLMDLRHPDFRESSRVSFEDQAPTME 41 Kv3.1MGQGDESERIVINVGGTRHQTYRSTLRTLPGTRLAWLAEPDAHSHFDYDPRADEFFFDRHPGVFAHILNYYRTGKLHCPADVCGPLYEEELAFWGIDETDVEPCCWMTYRQHRDAEEALDSFGGAPLDNSADDADADGPGDSGDGEDELEMTKRLALSDSPDGRPGGFWRRWQPRIWALFEDPYSSRYARYVAFASLFFILVSITTFCLETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTYIEGVCVVWFTFEFLMRVIFCPNKVEFIKNSLNIIDFVAILPFYLEVGLSGLSSKAAKDVLGFLRVVRFVRILRIFKLTRHFVGLRVLGHTLRASTNEFLLLIIFLALGVLIFATMIYYAERIGAQPNDPSASEHTHFKNIPIGFWWAVVTMTTLGYGDMYPQTWSGMLVGALCALAGVLTIAMPVPVIVNNFGMYYSLAMAKQKLPKKKKKHIPRPPQLGSPNYCKSVVNSPHHSTQSDTCPLAQEEILEINRAGRKPLRGMSI 42 Kv3.2MGKIESNERVILNVGGTRHETYRSTLKTLPGTRLALLASSEPQGDCLTAAGDKLQPLPPPLSPPPRPPPLSPVPSGCFEGGAGNCSSHGGNGGNGGSDHPGGGREFFFDRHPGVFAYVLNYYRTGKLHCPADVCGPLFEEELAFWGIDETDVEPCCWMTYRQHRDAEEALDIFETPDLIGGDPGDDEDLAAKRLGIEDAAGLGGPDGKSGRWRKLQPRMWALFEDPYSSRAARFIAFASLFFILVSITTFCLETHEAFNIVKNKTEPVINGTSPVLQYEIETDPALTYVEGVCVVWFTFEFLVRIVFSPNKLEFIKNLLNIIDFVAILPFYLEVGLSGLSSKAAKDVLGFLRVVRFVRILRIFKLTRHFVGLRVLGHTLRASTNEFLLLIIFLALGVLIFATMIYYAERVGAQPNDPSASEHTQFKNIPIGFWWAVVTMTTLGYGDMYPQTWSGMLVGALCALAGVLTIAMPVPVIVNNFGMYYSLAMAKQKLPRKRKKHIPPAPLASSPTFCKTELNMACNSTQSDTCLGKENRLLEHNRSVLSGDDSTGSEPPLSPPERLPIRRSSTRDKNRRGETCFLLTTGDYTCASDGGIRKASTLEPMESTAQTKGDTRPEAHWNCAH LLNFGCPTGSSFPTL 43 Kv3.3MLSSVCVSSFRGRQGASKQQPAPPPQPPESPPPPPLPPQQQQPAQPGPAASPAGPPAPRGPGDRRAEPCPGLPAAAMGRHGGGGGDSGKIVINVGGVRHETYRSTLRTLPGTRLAGLTEPEAAARFDYDPGADEFFFDRHPGVFAYVLNYYRTGKLHCPADVCGPLFEEELGFWGIDETDVEACCWMTYRQHRDAEEALDSFEAPDPAGAANAANAAGAHDGGLDDEAGAGGGGLDGAGGELKRLCFQDAGGGAGGPPGGAGGAGGTWWRRWQPRVWALFEDPYSSRAARYVAFASLFFILISITTFCLETHEGFIHISNKTVTQASPIPGAPPENITNVEVETEPFLTYVEGVCVVWFTFEFLMRITFCPDKVEFLKSSLNIIDCVAILPFYLEVGLSGLSSKAAKDVLGFLRVVRFVRILRIFKLTRHFVGLRVLGHTLRASTNEFLLLIIFLALGVLIFATMIYYAERIGADPDDILGSNHTYFKNIPIGFWWAVVTMTTLGYGDMYPKTWSGMLVGALCALAGVLTIAMPVPVIVNNFGMYYSLAMAKQKLPKKKNKHIPRPPQPGSPNYCKPDPPPPPPPHPFIHGSGGISPPPPITPPSMGVTVAGAYPAGPHTHPGLLRGGAGGLGIMGLPPLPAPGEPCPLAQEEVIEINRADPRPNGDPAAAALAHEDCPAIDQPAMSPEDKSPITPGSRGRYSRDRACFLLTDYAPSPDGSIRKATGAPPLPPQDWRKPGPPSFLPDLNANAAAWISP

In some cases, the one or more PV cell selective regulatory elementscomprise sequences of SEQ ID NOs: 1-32, a functional fragment or acombination thereof, or sequences comprising at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity thereto. In some cases,sequence identity is measured by BLAST. In some cases, such gene therapyis used to treat epilepsies, neurodegeneration, tauopathy, neuronalhypoexcitability, Dravet syndrome and/or Alzheimer's disease. In somecases, such gene therapy is used to treat epilepsy and/or seizuresassociated with Dravet syndrome and/or Alzheimer's disease. In somecases, treatment using a gene therapy described herein results inreduced seizure frequency and/or duration. In some cases, treatmentusing a gene therapy described herein results in increased formation offunctional sodium ion channels, functional potassium ion channels, orfunctional neurotransmitter regulatory in vivo.

In some cases, AAV serotypes 1, 8, and/or 9, or a hybrid thereof can beused with an expression cassettes described herein to target selectiveexpression in PV cells. In some cases, an expression cassette designedfor delivery by an AAV comprises a 5′ ITR, one or more cell-typeselective regulatory elements, an optional enhancer, an optional minimalpromoter, a transgene, optionally one or more introns, an optional polyAsignal, and a 3′ ITR. In some instances, an expression cassette cancontain a 5′ ITR, two cell-type selective REs, a basal promoter, atransgene, one or more post-transcriptional RNA regulatory elements, anda 3′ ITR.

An exemplary AAV expression cassette is illustrated in FIG. 7. In somecases, the expression cassette contains a 5′ AAV ITR, an enhancer (e.g.,PV cell selective enhancer or one or more combined regulatory elements),a promoter (e.g., one or more PV cell selective promoters or regulatoryelements), a transgene (e.g., SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1,KV3.2, KV3.3, STXBP1, or a DNA binding protein), a post-transcriptionalregulatory element, and a 3′ AAV ITR. The promoter can be PV cellselective, or a constitutive promoter. In some cases, the transgene is areporter gene, e.g., a coding sequence for eGFP, RFP, or a fluorescentmarker. In other cases, the transgene is a DNA binding protein thatmodulates gene expression.

In some cases, the transgene is a therapeutic transgene, e.g., a codingsequence for SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3,STXBP1, or a DNA binding protein, or a functional fragment or a sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity thereto. The post-transcriptional regulatoryelement can be any sequence which influences the expression of a proteinfrom an mRNA or stability of an RNA, for example, an intron, an internalribosome entry site (IRES), or a woodchuck hepatitis viruspost-transcriptional regulatory element. In some cases, thepost-transcriptional regulatory element is a combination of two or morepost-transcriptional regulatory elements.

The expression cassette can be designed for delivery by an optimizedtherapeutic retroviral vector, e.g., a lentiviral vector. The retroviralvector can be a lentiviral vector comprising a left (5′) LTR; sequenceswhich aid packaging and/or nuclear import of the virus, at least onecell-type selective regulatory element, optionally a lentiviral reverseresponse element (RRE); optionally a promoter or active portion thereof;a transgene operably linked to one or more regulatory elements;optionally an insulator; and a right (3′) retroviral LTR.

In some cases, the expression cassette comprises one or more cell-typeselective regulatory elements disclosed herein. In some cases, theexpression cassette comprises two or more regulatory elements combined.In some examples, the expression cassette comprises two or moreregulatory elements that are not combined, for example, a promoterupstream of the transgene and an enhancer or stability element locateddownstream of the transgene.

In some cases, the expression cassette contains a putative cell-typeselective regulatory element that has selective activity in a cell typeof interest, for example, a putative PV cell selective regulatoryelement. The expression cassette containing the putative regulatoryelement can be packaged in a viral vector and transfected into an animalmodel to assess the activity of the putative cell-type selectiveregulatory element. In some cases, a putative cell type selectiveregulatory element can be assessed in vitro or ex vivo by delivering avector containing the putative cell type selective regulatory elementinto a plurality of cells or cell types that include a target cell orcell type, and then comparing the cell type selective activity of theputative regulatory element to a control regulatory element, such as aconstitutive promoter or regulatory element, or a previously knownregulatory element.

In some cases, selective expression is used to selectively express atherapeutic moiety or a transgene in a cell-type of interest (ortissue-type of interest), such as PV neurons in the CNS. In some cases,a vector comprising a cell-type selective regulatory element operablylinked to a transgene results in an increased selective expression ofthe transgene in the cell-type of interest as compared to one or more(e.g., at least two, three, four, or five) other cells, cell types,tissues, or tissue types, or results in a preferred expression of thetransgene in the cell-type of interest as compared to one or more cellsor cell types, e.g., at least one, two, three, four, or five non-targetcell types.

Any known technique can be used to deliver the regulatory elements and atransgene, or compositions comprising regulatory elements and atransgene, to cells of interest (or a target cell or cell type) toconfer or induce in vitro, in vivo, or ex vivo expression of thetransgene in a cell-type selective manner.

The expression cassettes containing cell-type selective regulatoryelements of this disclosure further comprise one or more transgenes. Thetransgenes can be protein-coding genes. In some cases, the expressioncassette contains a transgene. The transgene can replace an absent ordefective gene, or compensate for deficient expression of a proteininside a cell. The transgene can be involved in a cell signalingpathway. In some cases, a transgene can encode a wild-type protein, afunctional fragment thereof, a variant or mutant protein having enhancedtherapeutic properties, e.g., enhanced activity. In some cases, thetransgene can encode a DNA binding protein comprising one or zinc fingeror a domain of dCas9, an ion channel, such as a potassium ion channel ora sodium ion channel, or a subunit thereof, a neurotransmitter factor ora neurotransmitter regulator. In some cases, a transgene can encode anion channel subunit, a variant, or a mutant thereof. In some cases, thetransgene is a DNA binding protein that comprises a DNA binding domainof a DNA binding protein or a DNA cleaving protein (e.g., a nuclease, arestriction enzyme, a recombinase, etc.) wherein the DNA cleaving domainor nuclease domain has been deactivated, e.g., a nuclease-deactivatedCas (dCas), a deactivated transcription activator-like effectornuclease, or a nuclease-deactivated zinc finger protein. In some cases,the DNA binding domain is linked to a transcriptional modulating domain(e.g., a transcriptional activator or repressor domain). In some cases,the transgene comprises a gene editing protein, e.g., a Cas protein,Cas9.

The regulatory elements disclosed herein can be located at any positionwithin an expression vector or cassette. For example, the regulatoryelements can be positioned upstream of an enhancer, downstream of anenhancer but upstream of a promoter, within the 5′ UTR of a transgene,within an intron in the transgene, in the 3′ UTR of the transgene, ordownstream of the transgene. In some cases, one or more regulatoryelements are positioned upstream or downstream of the operably linkedtransgene.

In some examples, a regulatory element of this disclosure results inselective expression of an operably linked transgene at a level that isat least 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.5, or 3 IU/ml in a target cell type (e.g., PV cells) as measured byELISA. In some cases, a regulatory element's ability to increasetransgene expression can be assessed in a mouse wherein the total amountof transgene expression in the whole mouse and/or the total number ofcell types or tissue types having transgene expression are measured.

When assessing the activity of an expression cassette or vector, theactivity or expression can be represented as an activity or expressionlevel per unit dose, or normalized to a dose of expression cassette orvector administered or delivered to a cell, mouse, or a subject. In somecases, expression or activity of a transgene is normalized to an amountof plasmid or DNA (e.g., μg/kg per mouse), or viral particles (e.g.,normalized to an amount of genome copies/kg per mouse or subject) usedto allow comparison across different expression vectors or cassetteswith or without a regulatory element. For example, when assessing aregulatory element's activity in a mouse, selective expression oractivity in PV cells assayed can be normalized to a dose of about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 10or 720 μg of expression vector, cassette, or plasmid per mouse. In somecases, the expression level or activity can be normalized to 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, or 10¹⁵ gc/kg of viral particles containing anexpression vector or cassette as disclosed herein per mouse.

In some aspects, an expression cassette comprises one or more regulatoryelements (e.g., any one or more of SEQ ID NOs: 1-32, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto) disclosed herein operably linked to a transgene to result incell-type selective expression, or preferential expression, of thetransgene in a target cell type over at least one, at least two, atleast three, at least four, at least five, or more than five non-targetcell types. In some cases, an expression cassette comprises one or moreregulatory elements operably linked to a transgene to result incell-type selective expression or preferential expression in a targetcell subtype over at least one, at least two, at least three, at leastfour, at least five, or more than five non-target subtypes, or all otherknown subtypes of the cell. In some cases, the transgene is any one ofSCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, and STXBP1, or afunctional fragment thereof, or sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% sequence identitythereto. In some cases the transgene is a DNA binding protein thatmodulates an endogenous gene (e.g., an endogenous SCN1A, SNC2A, SNC8A,SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, or STXBP1). In some cases, thetransgene is any one of SEQ ID NOs: 36-43, or a functional fragmentthereof, or sequences having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto. In some cases,the transgene is a transcriptional modulator. In some cases, thetransgene is a DNA binding protein that comprises a DNA binding domainof a DNA binding protein or a DNA cleaving protein (e.g., a nuclease, arestriction enzyme, a recombinase, etc.) wherein the DNA cleaving domainor nuclease domain has been deactivated, e.g., a nuclease-deactivatedCas (dCas), a deactivated transcription activator-like effectornuclease, or a nuclease-deactivated zinc finger protein. In some cases,the DNA binding domain is linked to a transcriptional modulating domain(e.g., a transcriptional activator or repressor domain). In some cases,the transgene is a gene editing protein, such as a Cas family protein,Cas9, a zinc finger nuclease, a zinc finger nuclease, or a transcriptionactivator-like effector nuclease. In some cases, the transgene is areporter gene or a fluorescent marker. In some cases, an expressioncassette disclosed herein is in a viral vector. In some cases, anexpression cassette disclosed herein is packaged in an rAAV, such asrAAV9 or rAAVDJ. In some cases, an expression cassette disclosed hereinis delivered into a cell as a gene therapy. In some cases, a genetherapy disclosed herein is delivered into a subject, preferably a humanor a mammal. In some cases, an expression cassette disclosed herein isused to treat a neurological condition or disease, such as epilepsy, aneurodegenerative disease, tauopathy, neuronal hypoexcitability, Dravetsyndrome or Alzheimer's disease.

In some cases, an expression cassette (e.g., gene therapy, viral vector,vector, or plasmid) comprises any one or more of SEQ ID NOs: 1-32, or afunctional fragment or a combination thereof, or sequences having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto, operably linked to a transgene. In somecases, such combined regulatory elements are linked using a linker of1-50 nucleotides. In some cases, such combined regulatory elements arenot linked. In some cases, two or more regulatory elements are locatedupstream and/or downstream of the promoter. In some cases, two or moreregulatory elements are located upstream and/or downstream of thetransgene.

In some cases, an expression cassette comprises one or more regulatoryelements disclosed herein, when operably linked to any transgene (e.g.,a reporter transgene or a therapeutic transgene), drives selectiveexpression or preferential expression in at least one target cell typeat a level that is statistically significantly higher than theexpression driven by CAG, EF1α, a constitutive promoter, or anon-selective regulatory element when operably linked to the sametransgene, or by the same construct without the regulatory elements. Insome cases, statistically significantly higher means the regulatoryelements drive selective expression in the target cell type at a levelthat is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, or 100 times the expression level by CAG, EF1α, a constitutivepromoter, or a non-selective regulatory element when operably linked tothe same transgene in the target cell type, or by the same constructwithout the regulatory elements. In some cases, such cell-type selectiveexpression is assayed using a co-localization assay as described herein.

In other aspects, an expression cassette comprising one or moreregulatory elements disclosed herein operably linked to any transgenedisclosed herein results in selective expression or preferentialexpression of the transgene in a target cell type over at least one, atleast two, at least three, at least four, at least five, or more thanfive non-target cell types or non-target subtypes.

In some instances, the target cell type is a PV cell. In some cases, thenon-target cell subtypes are at least one, at least two, at least three,or at least four of the non-PV GABAergic subtypes disclosed herein. Insome cases, an expression cassette comprising a regulatory elementdisclosed herein is selective for PV cells over all non-PV GABAergiccells or all non-PV CNS cells. In some cases, cell-type selectivity ismeasured according to a co-localization assay disclosed herein. In somecases, cell-type selectivity is measured using a mouse that expressesCre in the target cell type.

Parvalbumin (PV) Neurons

GABAergic neurons produce gamma aminobutyric acid (GABA), the maininhibitory neurotransmitter in the CNS. GABA is important for reducingneural excitability throughout the nervous system. GABA acts atinhibitory synapses by binding specific transmembrane receptors andcausing the opening of ion channels which negatively change the membranepolarization. This generally results in hyperpolarization of the celland increases the signal required to trigger an action potential.Defects in GABAergic neurons can result in an imbalance betweenexcitatory and inhibitory signaling, and have been implicated in manyneurological diseases, including Dravet syndrome, epilepsy,neurodegeneration, tauopathies and Alzheimer's disease. Otherneurological conditions or diseases implicated include a psychiatricdisorder (e.g., schizophrenia, obsessive compulsive disorder, addiction,depression, anxiety, psychosis); an autism spectrum disorder (e.g.,Fragile X syndrome, Rett syndrome); epilepsy (e.g., chronic traumaticencephalopathy, generalized epilepsy with febrile seizures plus (GEFS+),epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy,tuberous sclerosis); and/or neurodegeneration (e.g., Alzheimer'sdisease, Parkinson's disease). In some cases, the neurological conditionor disease is any seizure and/or epilepsy related condition or diseasewherein PV neurons are implicated.

Parvalbumin is a calcium-binding protein, which is expressed in about40% of total GABAergic interneurons in the somatosensory cortex. Withinthe CNS, PV cells are generally considered GABAergic cells. Variousstudies have also identified GABAergic cells to include distinctsubtypes of cells, including cells that express PV, SOM, CR, CCK, NPY,VIP, or a combination thereof.

PV neurons are particularly relevant for various neurological diseasesor conditions, such as Dravet syndrome, Alzheimer's disease, epilepsy,neurodegeneration, tauopathies and/or seizures. In some cases, a PVneuron-associated neurological condition or disease is a psychiatricdisorder (e.g., schizophrenia, obsessive compulsive disorder, addiction,depression, anxiety, psychosis); an autism spectrum disorder (e.g.,Fragile X syndrome, Rett syndrome); epilepsy (e.g., chronic traumaticencephalopathy, generalized epilepsy with febrile seizures plus (GEFS+),epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy,tuberous sclerosis); or neurodegeneration (e.g., Alzheimer's disease,Parkinson's disease). In some cases, the neurological condition ordisease is any seizure and/or epilepsy related condition or diseasewherein PV neurons are implicated. In various aspects, the target cellis PV cells in the CNS, or GABAergic cells that express PV.

In various aspects, PV-expressing interneurons are also called basketcells, which can be further subdivided by size of the cell body (e.g.,large basket cell, small basket cell, and nest basket cell), anddendritic and axonal projection. Physiologically, PV-expressing basketcells are often fast-spiking (FS), characterized by a high-frequencytrain of action potentials (APs) with little adaptation. It is widelyaccepted that PV basket neurons innervate the soma and proximaldendrites of excitatory pyramidal neurons. Feedforward inhibitionmediated through FS PV-expressing basket neurons can be found in severalcortical networks including thalamocortical, translaminar, andinterareal circuits. FS PV basket neurons strongly inhibit neighboringexcitatory pyramidal neurons. It has been shown that PV basket neuronsand pyramidal neurons that share common excitatory inputs tend to bereciprocally connected (feedback inhibition). These connections canserve to regulate the precise time window in which the excitatoryneurons can generate spikes in response to excitatory drives. Inaddition, thalamocortical and intracortical excitatory inputs onto FS PVbasket neurons are depressed by high frequency stimulation, whichmediates activity-dependent feedforward inhibition. PV-expressing basketcells also innervate other interneurons including other basket cells,and are electrically coupled with each other through gap junctions. Ithas been proposed that this feature may help to generate and maintaincortical network synchronization and oscillation.

In some cases, one or more regulatory elements disclosed herein resultin increased selectivity in gene expression in PV neurons as compared toat least one, at least two, at least three, at least four, or at leastfive non-PV expressing neurons. In some cases, non-PV cells include allnon-PV GABAergic cells. In some cases, non-PV GABAergic neurons include,but are not limited to, calretinin (CR), somatostatin (SOM),cholecystokinin (CCK), CR+SOM, CR+neuropeptide Y (NPY),CR+vasointestinal polypeptide (VIP), SOM+NPY, SOM+VIP, VIP+cholineacetyltransferase (ChAT), CCK+NPY, CR+SOM+NPY, and CR+SOM+VIP expressingcells.

In some cases, any one of CAG, EF1α, a constitutive promoter (e.g.,SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory elementthat drives gene expression in a non-cell type selective manner can beused for comparison with PV selective regulatory elements, or any celltype selective regulatory elements, disclosed herein. In some cases, aregulatory element that results in selective expression in PV cells at alevel above the expression of a gene operably linked to CAG or EF1αcontrol is indicative of selectivity to PV cells. In some cases, aregulatory element disclosed herein shows selective gene expression inPV cells that is at least 2%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% higher than the PV expression level from a transgene that isoperably linked to CAG, EF1α, a constitutive promoter (e.g., SV40, CMV,UBC, PGK, and CBA), or a non-selective regulatory element (e.g., SEQ IDNO: 34, or a functional fragment thereof, or a sequence having at least80%, at least 85%, at least 90%, at least 95%, or at least 99% sequenceidentity thereto), and as measured in a co-localization assay. In somecases, a regulatory element disclosed herein shows a selective geneexpression in PV cells that is at least 1.5 fold, at least 2 fold, atleast 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, atleast 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold theexpression level under CAG, EF1α, a constitutive promoter (e.g., SV40,CMV, UBC, PGK, and CBA), or a non-selective regulatory element (e.g.,SEQ ID NO: 34, or a functional fragment thereof, or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto) and as measured in a co-localization assaydescribed herein.

In preferred cases, one or more regulatory elements described hereinselectively drive expression of a transgene in a GABAergic cell, such asa GABAergic cell that expresses parvalbumin over at least one other CNScell type (e.g., at least two, at least three, at least four, at leastfive non-PV cells, or two or more, three or more, or four or more non-PVcells and/or non-PV GABAergic neurons).

In some cases, a target cell type is a GABAergic neuron that expressesparvalbumin, or PV cells.

One way of selectively expressing a transgene within a subpopulation ofcells in the brain is to use a viral vector comprising a transgeneoperably linked to a cell-type selective regulatory element, or aregulatory element that is selective (or has selective activity) in thesubpopulation of cells in the brain, e.g., PV cells. A viral vector canbe selected to have high infectivity without selectivity for aparticular cell type, while the regulatory element confers selectivity.For example, a cell-type selective regulatory element can driveexpression of a transgene in PV neurons and not in other neurons.

In some cases, the present disclosure involves the use of regulatoryelements (i.e., PV cell selective regulatory elements) that selectivelydrive expression in PV neurons.

GABAergic cells are inhibitory neurons which produce gamma-aminobutyricacid. GABAergic cells can be identified by the expression of glutamicacid decarboxylase 2 (GAD2). Other markers of GABAergic cells includeGAD1, NKX2.1, DLX1, DLX5, SST, PV and VIP.

In some instances, a non-PV CNS cell is an excitatory neuron, adopaminergic neuron, an astrocyte, a microglia, a motor neuron or avascular cell. In some instances, a non-GABAergic neuron is a cell thatdoes not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST andVIP. In some instances, a non-PV neuron is a GABAergic neuron that doesnot express parvalbumin. In some instances, other CNS cells refer to CNScell types that have never expressed any of PV, GAD2, GAD1, NKX2.1,DLX1, DLX5, SST and VIP.

In some instances, a regulatory element disclosed herein is selectivefor a PV expressing cell over at least one, two, three, four, five, ormore than five non-PV CNS cell-types. In some cases, non-PV cell typesinclude non-PV GABAergic cells. In some cases, the cell type of interestis a PV cell. In some cases, REs selective for PV cells are referred toas PV cell selective regulatory elements.

In some cases, the PV cell selective regulatory elements disclosedherein include the sequences of SEQ ID NOs: 1-32, or any combinationthereof.

In some cases, one or more PV cell selective regulatory elements or oneor more regulatory elements disclosed herein are used to increaseexpression of a transgene in PV-expressing cells by at least 2, 5, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold as compared toexpression without the regulatory element. In some cases, a RE in anexpression cassette increases gene expression by at least 1.5%, 2%, 5%,10%, 15%, 20%, or 50%, or more than 1.5%, 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% as compared to expression without the regulatory elements. In somecases, compositions and methods of use thereof comprise expressioncassettes containing one or more regulatory elements that result in a10-500% increase in transgene expression, e.g., expression of SCN1A,SNC2A, SNC8A, SCN1B, SCN2B, KCNC1 (also known as KV3.1), KCNC3 (alsoknown as KV3.3), STXBP1, a DNA binding protein, or a variant orfunctional fragment thereof, or a protein thereof, as compared to thelevel without the regulatory elements or as compared to a non-selectiveregulatory element (e.g., CAG, EF1α, a constitutive promoter, or SEQ IDNO: 34, or a functional fragment thereof, or a sequence having at least80%, at least 85%, at least 90%, at least 95%, or at least 99% sequenceidentity thereto). In some cases, the increase in gene expression and/orprotein level of any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KCNC1,KCNC3, and STXBP1 is 1.5-5%, 5%-10%, 10-15%, 15-20%, 20-30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-150%, 150-200%,250-300%, 300-350%, 350-400%, 400-450%, 450-500%, or 1.5-20%, 20%-50%,50%-100%, 100-200%, 200-300%, 300-400%, or 400-500% as compared to thelevel without the expression cassette or regulatory elements. In somecases, such gene or protein expression is selective in PV cells ascompared to an expression cassette comprising a control (e.g., CAG,EF1α, a constitutive promoter (e.g., SV40, CMV, UBC, PGK, and CBA), or anon-selective regulatory element) or a non-cell type selectiveregulatory element (e.g., SEQ ID NO: 34, or a functional fragmentthereof, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto).

In some cases, selectivity of expression in PV cells can be calculatedby dividing the number of cells that express both PV and eGFP (thetransgene operably linked to one or more regulatory elements) by thetotal number of cells that express eGFP, and multiplying by 100 toconvert into a percentage. PV cell selective regulatory elements asdescribed herein can be highly selective for expression in PV cells. Forexample, PV cell selective regulatory elements or one or more regulatoryelements disclosed herein can exhibit about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orgreater than about 99% selectivity for PV neurons.

In some cases, a PV cell selective regulatory element or any regulatoryelement disclosed herein confers selectivity in expressing a transgenein PV neurons at a level that is statistically higher than a controlregulatory element, e.g., EF1α or a previously known regulatory element.In some instances, the statistical difference between a PV cellselective regulatory element and a control regulatory element is atleast 2 fold, 5 fold, 10 fold, 20 fold, or more than 2 fold difference,or more than 5 fold, 10 fold, or 20 fold difference as determined by anyone of the methods described herein, such as a co-localization assay.

The present disclosure includes regulatory elements that are selectivefor PV cells. These PV cell selective REs or any cell type selective REsare preferably short, preferably less than about 1100 base pairs, 1000bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, orless than about 110 bp. The PV cell selective REs or any cell typeselective REs can be between 1050 bp and 100 bp, between 100 bp and 500bp, or between 500 bp and 1050 bp. Some examples of PV cell selectiveregulatory elements are provided by SEQ ID NOs 1-32, or a functionalfragment or combination thereof. Other PV cell selective regulatoryelements contemplated by the present disclosure include sequences havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity to any of these sequences described herein, or a partor fragment of one of the sequences described herein.

In some cases, a PV cell selective regulatory element or any regulatoryelement disclosed herein has at least about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to asequence described herein, or a fragment of a sequence described herein.In some cases, a PV cell selective regulatory element has at least about80% identity to at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%of a sequence described herein, or a functional fragment thereof.

In some cases, a PV cell selective regulatory element comprises at least80% identity to any one or more of SEQ ID NOs: 1-32, or a functionalfragment or a combination thereof, or sequences having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% sequence identitythereto. In some cases, a PV cell selective regulatory element has 90%identity to 50% or more of a sequence of SEQ ID NOs: 1-22. In someinstances, a PV-selective regulatory element is a functional fragment ofany of SEQ ID NOs: 1-32 or a combination thereof. In some cases, thefunctional fragment is able to selectively express a transgene in PVcells with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orgreater than 95% selectivity of expression in PV cells.

In some cases, two or more PV cell selective regulatory elements of thisdisclosure or any two or more regulatory elements disclosed herein arecombined to form combination regulatory elements. In some instances, twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, or ten or more PV cell selectiveregulatory elements, or a plurality of regulatory elements disclosedherein, are combined. For example, SEQ ID NO: 31 is a combination of SEQID NOs 1 and 23-29. As another example, SEQ ID NO: 32 is a combinationof SEQ ID NO: 8 and SEQ ID NO: 23-29. In some cases, fragments of two ormore PV-selective regulatory elements can be combined to form acombination regulatory element. For example, 50% of SEQ ID NO: 1 can becombined with 30% of SEQ ID NO: 8 and 90% of SEQ ID NO: 30 to form acombination regulatory element.

In some cases, one or more PV cell selective regulatory elements of thisdisclosure or any one or more regulatory elements disclosed hereinselectively express an operably linked transgene in PV neurons ascompared to one or more other CNS cell types. This selective expressioncan be quantified by counting the number of PV neurons which expressdetectable levels of the linked transgene as a percentage of the totalnumber of cells expression the transgene, including the number of non-PVneurons which express the transgene. In other words, selectivity of a PVregulatory element in a particular cell type or target cell can bedetermined by measuring and/or comparing the number of PV neurons (ortarget cells) expressing the transgene that is operably linked to theregulatory element relative to the number of non-target cell types thatexpress the transgene (or the total number of cells expressing thetransgene).

In some cases, PV cell selective regulatory elements can exhibit about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or greater than about 99% selectivity for PVneurons, PV neurons in the CNS, or GABAergic neurons that also expressPV. In some cases, one or more regulatory elements of this disclosurecan exhibit about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% selectivityfor PV neurons as compared to CAG or EF1α or non-cell-type selectiveelement, or as compared to non-PV CNS cells, or as compared to at leastone, at least two, at least three, at least four, or at least five othernon-PV GABAergic neuronal sub-types in the CNS.

In some cases, a PV selective regulatory element confers selectivity inexpressing a transgene in PV neurons at a level that is statisticallyhigher than a control regulatory element, e.g., CAG, EF1α, aconstitutive promoter (e.g., SV40, CMV, UBC, PGK, and CBA), anon-selective regulatory element, or a previously known regulatoryelement. In some instances, the statistical difference between a PV cellselective RE and a control element is at least 2 fold, 5 fold, 10 fold,20 fold, or more than 2 fold difference, or more than 5 fold, 10 fold,or 20 fold difference as determined by any one of the methods describedherein. In some cases, the selectivity in PV is measured using aco-localization assay as described herein.

In some aspects, the cell-type selective regulatory elements describedherein are useful for selectively modulating expression of a transgenein a CNS cell type compared to other CNS cell types. For example, thecell-type selective regulatory elements described herein can be usefulfor selectively modulating expression of a transgene in PV cells overother CNS cells, including other types of neurons. For gene therapy,selective expression of a transgene in a target cell type and/orminimized expression of the transgene in a non-target cell type can bedesired. Expression of the transgene in an unintended cell-type (e.g.,non-target cell type) can result in an adverse effect to the subject.Expression of the transgene in an unintended cell-type can counteractthe therapeutic effect of the transgene in the intended cell type. Forexample, a transgene intended for expression in PV cells can have anegative effect for the subject if expressed in glutamatergic neurons.The cell-type selective regulatory elements described herein can be usedin expression cassettes to ensure appropriate expression of a transgeneand/or to reduce off-target effects of gene therapy.

The cell-type selective regulatory elements herein can be used in geneexpression cassettes whereby they are operably linked to one or moretransgenes. Such gene expression cassettes are used to delivertransgenes into cells for expression. The expression cassette cancontain a cell-type selective regulatory element as described herein, acombination of cell-type selective regulatory elements, or a fragment ofa cell-type selective regulatory element as described herein operablylinked to a transgene.

Preferably, the expression cassettes herein include one or morecell-type selective regulatory elements operably linked to a transgene,whereby the two do not function together in their endogenous context invivo. For example, a transgene for sodium ion channel beta subunit, suchas SCN1B, can be operably linked to one or more regulatory elements thatdo not function in the same context in vivo, or do not detectably driveexpression of SCN1B endogenously. Similarly, a nucleic acid cassette caninclude a neurotransmitter regulator, such as STXBP1, operably linked toa regulatory element that does not function in the same contextendogenously or in vivo, or is not in the same open reading frame, or isnot on the same human chromosome, or does not detectably driveexpression of STXBP1 in vivo. In some cases, a cell-type selectiveregulatory element is linked to a transgene, wherein the cell-typeselective regulatory element does not regulate the endogenous genecorresponding to the transgene in vivo.

In some aspects, cell-type selective regulatory elements disclosedherein are derived from sequences isolated from a human chromosomallocus different from a locus of a native gene corresponding to thetransgene. Thus, in some instances, an expression cassette comprisescell-type selective regulatory element(s) having a sequence derived froma chromosome different from the chromosome corresponding to thetransgene on the same cassette. In other instances, regulatoryelement(s) of the disclosure and a transgene that are operably linked inan expression cassette are derived from sequences located more than 20kb apart in the human genome, or at distal genomic locations. When twoor more human derived regulatory elements are utilized on an expressioncassette, the two or more regulatory elements can have sequences locatedmore than 5 kb apart, more than 10 kb apart, more than 15 kb apart, ormore than 20 kb apart in the human genome, or wherein the two or moreregulatory elements do not interact with each other naturally in thegenome.

In some cases, an expression cassette comprising PV-selective regulatoryelements can exclude known sequences derived from hSyn1 or GAD2 promotersequences. In some instances, the PV-selective regulatory elements donot comprise the full promoter sequence of any one of the GAD2, GAD1,SYN1, NKX2.1, DLX1, DLX5, SST and VIP promoters. In some instances, thePV-selective regulatory elements do not comprise more than 500contiguous base pairs of sequence derived from the promoter or one ormore of GAD2, GAD1, SYN1, NKX2.1, DLX1, DLX5, SST and VIP. In someinstances, the PV-selective regulatory elements do not comprisesequences which are within 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8kb, 9 kb, or 10 kb of the transcription start site of any one of GAD2,SYN1, NKX2.1, DLX1, DLX5, SST, and VIP.

In some cases, a transgene is useful to treat a disease associated witha specific cell type of interest. In some cases, a cell type of interestis a neuron, an inhibitory neuron, a GABAergic neuron, or a PV neuron.In some cases, a transgene is any one or more of SCN1A, SNC2A, SNC8A,SCN1B, SCN2B, KV3.1, KV3.3, and STXBP1. In some cases, a transgene is aDNA binding protein that modulates expression of a gene (e.g., atranscriptional activator or a transcriptional repressor that modulatesexpression of an endogenous gene). In some cases, a transgene is a geneediting protein, such as a zinc finger nuclease, a transcriptionactivator-like effector nuclease, a Cas family protein. In some cases, atransgene is a reporter gene or a detectable marker, such as eGFP,tdTomato, or RFP. In some cases, a transgene is a Cas protein, such asCas9.

Transgenes useful to treat a condition associated with PV neuron cellscan be incorporated in a vector, nucleic acid cassette, or method asdescribed herein. Transgenes used herein generally do not containintrons, or do not contain more than one intron. A transgene can beobtained from a cDNA sequence rather than from genomic sequence. In somecases, transgenes can contain some, or all, of their endogenous introns.In some examples, such a transgene encodes for a DNA binding domain oran ion channel. Examples of DNA binding domains that can be encoded forin the expression cassettes of this disclosure include zinc fingers,Cas9, a Cas family protein, dCas9, a dCas family protein or atranscriptional activator like effector (TALE). In some cases, thetransgene is a DNA binding protein that comprises a DNA binding domainof a DNA binding protein or a DNA cleaving protein (e.g., a nuclease, arestriction enzyme, a recombinase, etc.) wherein the DNA cleaving domainor nuclease domain has been deactivated, e.g., a nuclease-deactivatedCas (dCas), a deactivated transcription activator-like effectornuclease, or a nuclease-deactivated zinc finger protein. In some cases,the DNA binding domain is linked to a transcriptional modulating domain(e.g., a transcriptional activator or repressor domain). In some cases,the transgene comprises a gene editing protein, e.g., a Cas protein,Cas9. In some cases, the transgene is a subunit or a component of an ionchannel or a membrane protein, or a gene associated with a neurologicalcondition or disease disclosed herein. Examples of ion channeltransgenes which can be used in the expression cassettes of thisdisclosure include voltage gated and ligand gated ion channels. Voltagegated ion channels include sodium channels, calcium channels, potassiumchannels, and proton channels. In some instances, the transgene codesfor a subunit of a voltage gated sodium channel. Examples of voltagegated sodium channel subunits include SCN1B (NM_001037.4), SCN1A(NM_001165963.1), and SCN2B, (NM_004588.4).

In some instances, the transgene codes for a subunit of a voltage gatedpotassium channel. Examples of voltage gated sodium channel subunitsinclude KCNC1 (NM_001112741.1), and KCNC3 (NM_004977.2). In some cases,a transgene is any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B,KV3.1, KV3.3, STXBP1, a variant and a functional fragment thereof. Insome cases, a transgene is a sequence having at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.5% sequence identity to any one of SCN1A, SNC2A, SNC8A, SCN1B,SCN2B, KV3.1, KV3.3, STXBP1, a variant or a functional fragment thereof.In some cases, such sequence identity is measured using BLAST.

In some cases, an expression cassette disclosed herein comprises one ormore PV-selective regulatory elements or one or more regulatory elementsof this disclosure operably linked to a transgene having at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% sequence identity to a sequence encodingany one of SEQ ID NOs: 37-43, or a functional fragment or variantthereof, or the GenBank sequences corresponding to SEQ ID NOs: 37-43. Insome cases, an expression cassette disclosed herein comprises atransgene having a sequence according to (i) a sequence of SEQ ID NOs:37-43, or (ii) a functional fragment thereof, or (iii) a sequence havingat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% sequence identity to (i) or(ii).

In some examples, the transgene is a neurotransmitter regulator, or avariant or functional fragment thereof. A neurotransmitter regulator maybe involved in regulating production or release of a neurotransmitter inthe CNS. For example, a neurotransmitter regulator may assist withsynaptic fusion to release neurotransmitters. An example of aneurotransmitter regulator is STXBP1 (NM_001032221.3) or a functionalfragment thereof, or a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto. Thetransgene may also be a subunit of a neurotransmitter regulator.

In some case, an expression cassette of this disclosure can contain anAAV2 5′ ITR, a PV-selective enhancer, a PV-selective promoter, or acombination of one or more PV-selective promoters and enhancers, a cDNAof SCN1B, a WPRE, a hGH polyA signal, PV-selective regulatory element,and an AAV2 3′ITR. In one example, the expression cassette comprises anAmpR promoter, and AmpR coding sequence, a bacterial origin ofreplication, an AAV2 ITR, SEQ ID NO: 8, SEQ ID NOs: 23-29, a transgene(coding sequence, a WPRE, a human growth hormone poly A signal, a AAV2ITR, and an f1 origin.

As another example, an expression cassette of this disclosure cancontain an AAV2 5′ ITR, an enhancer, a promoter, a transcriptionalactivator of the endogenous SCN1A gene, a WPRE, a hGH polyA signal, aregulatory element, and an AAV2 3′ITR. In some cases, an expressioncassette comprises AAV2 5′ ITR, a promoter, an intronic element,transcriptional modifier, synthetic polyA, and an AAV2 3′ ITR. In somecases, an expression cassette of this disclosure can contain an AAV2 5′ITR, a PV-selective enhancer, a PV-selective promoter, a sequenceencoding a transcriptional activator of SCN1A or SCN1B, a WPRE, a hGHpolyA signal, a PV-selective regulatory element or any of the regulatoryelements disclosed herein, and an AAV2 3′ITR.

An expression cassette comprising one or more regulatory element of thisdisclosure can be used to treat a medical condition. In some cases, anexpression cassette containing a regulatory element of this disclosureis used to treat a neurological condition or a neurodegenerativecondition. The neurological condition can be caused by a known geneticevent or may have an unknown cause.

The neurological condition can be a disease associated with PV neurons.The neurological condition can be a disease associated with inhibitoryneurons, such as PV neurons. Diseases or conditions associated with PVneurons can be treated by delivering an expression cassette carrying atransgene and one or more PV cell-selective regulatory elements or anyof the regulatory elements as described herein to a cell in vivo. Insome aspects, expression cassettes comprising a transgene operablylinked to one or more PV cell-selective regulatory elements or any ofthe regulatory elements disclosed herein can be used to treat Dravetsyndrome, Alzheimer's disease, epilepsy, a neurodegenerative disorder,tauopathy, neuronal hypoexcitability and/or seizures. In some cases, anexpression cassette of this disclosure is used to treat a psychiatricdisorder (e.g., schizophrenia, obsessive compulsive disorder, addiction,depression, anxiety, psychosis); an autism spectrum disorder (e.g.,Fragile X syndrome, Rett syndrome); epilepsy (e.g., Dravet syndrome,chronic traumatic encephalopathy, generalized epilepsy with febrileseizures plus (GEFS+), epileptic encephalopathy, temporal lobe epilepsy,focal epilepsy, tuberous sclerosis); and/or neurodegeneration (e.g.,Alzheimer's disease, Parkinson's disease). In some cases, theneurological condition or disease is any seizure and/or epilepsy relatedcondition or disease wherein PV neurons are implicated.

Majority of Dravet syndrome cases is associated with mutations in theSCN1A and/or SCN2A genes. Mutations or abnormalities in SCN1A has alsobeen associated with seizure disorders, epilepsy, autism, familialhemiplegic migraine type 3 (FHM3), genetic epilepsy with febrileseizures plus (GEFS+), and effectiveness of certain anti-seizuremedications. For instance, ICS5N+5G>A mutation in SCN1A is associatedwith the maximum safe amount (dose) of the anti-seizure drugs phenytoinand carbamazepine.

In some Alzheimer's patients, production of amyloid β (Aβ) involvingmany peptides and proteases that can affect excitability of neurons,causing seizures and downregulation of the Nav1.1 sodium channel in PVneurons.

Diseases associated with dysfunctional PV neurons such as those due toloss of function mutations in SCN1A or Nav1.1 include: Dravet syndrome,Ohtahara syndrome, epilepsy, early infantile epileptic encephalopathy 6(EIEE6), familial febrile seizures 3A (FEB3A), intractable childhoodepilepsy with generalized tonic-clonic seizures (ICEGTC), migraine,familial hemiplegic 3 (FHM3), Panayiotopoulos syndrome, familial atrialfibrillation 13 (ATFB13), generalized epilepsy with febrile seizuresplus type 1 (gefs+ type 1), Brugada syndrome, nonspecific cardiacconduction defect, generalized epilepsy with febrile seizures plus,benign familial infantile seizures, early infantile epilepticencephalopathy11 (EIEE11), benign familial infantile epilepsy,neurodegeneration, tauopathies and Alzheimer's disease. In some cases,the neurological condition is Dravet syndrome. Dravet syndrome isassociated with mutations in the SCN1A and/or SCN2A genes. In somecases, one or more regulatory elements of this disclosure are used in agene therapy or an expression cassette to treat a neurological conditionor disease associated with PV neurons, e.g., a psychiatric disorder(e.g., schizophrenia, obsessive compulsive disorder, addiction,depression, anxiety, psychosis); an autism spectrum disorder (e.g.,Fragile X syndrome, Rett syndrome); epilepsy (e.g., Dravet syndrome,chronic traumatic encephalopathy, generalized epilepsy with febrileseizures plus (GEFS+), epileptic encephalopathy, temporal lobe epilepsy,focal epilepsy, tuberous sclerosis); or neurodegeneration (e.g.,Alzheimer's disease, Parkinson's disease). In some cases, one or moreregulatory elements of this disclosure (e.g., PV neuron selectiveregulatory elements) are used to treat Dravet syndrome and/orAlzheimer's disease (e.g., in an expression cassette, a vector, or agene therapy). In some cases, the neurological condition or disease isany seizure and/or epilepsy related condition or disease wherein PVneurons are implicated.

Methods and compositions of this disclosure can be used to treat asubject who has been diagnosed with a disease, for example, aneurological or neurodegenerative disease. The subject can be a patientsuffering from a form of epilepsy. In some instances, the subject is apatient with Dravet syndrome. The subject can be a patient sufferingfrom a neurodegenerative disease, for example, a patient withAlzheimer's disease. In some instances, epilepsy, encephalopathy, and/orseizures are associated with a genetic mutation in SCN8A. In some cases,a genetic mutation in SCN8A can give rise to epilepsy syndromes, e.g.,Dravet syndrome. In some instances, a genetic mutation in STXBP1 isassociated with encephalopathy with epilepsy, characterized by recurrentseizures.

In some instances, a subject treated with one or more compositionsdescribed herein is one diagnosed with a mutation or genetic aberrationin an ion channel or a neurotransmitter regulator (e.g., a syntaxinbinding protein). Examples of such mutations include mutations in SCN1A,SNC2A, SNC8A, SCN1B, SCN2B, KCNC1, KCNC3, and/or STXBP1, or combinationthereof. The expression cassette containing a cell-type selectiveregulatory element as described herein can be delivered to a subject totreat or prevent a disease with symptoms associated with a specific celltype. For example, an expression cassette comprising a transgeneoperably linked to one or more PV cell selective regulatory element isdelivered to a subject who has symptoms, or is at risk of developingsymptoms, associated with PV neurons.

In some cases, the treatment can be administered to a subject with, orat risk of developing, Dravet syndrome. Symptoms associated with Dravetsyndrome include seizures, memory defects, developmental delay, poormuscle tone and/or cognitive problems. Treatment with an expressioncassette of this disclosure can result in an improvement of one or moresymptoms, such as a reduction in number, duration, and/or intensity ofseizures. Administration of a gene therapy as described herein to asubject at risk of developing Dravet syndrome can prevent thedevelopment of or slow the progression of one or more symptoms.

In another example the treatment may be administered to a subjectsuffering from Alzheimer's disease. Symptoms associated with Alzheimer'sdisease include short term memory loss, cognitive difficulties,seizures, and difficulties with language, executive functions,perception (agnosia), and execution of movements (apraxia). Treatmentwith an expression cassette of this disclosure can result in animprovement of one or more Alzheimer's disease symptoms, such as areduction in progression of memory loss, or the prevention of one ormore symptoms. In some cases, the treatment can result in a correctionof high gamma power brain activity. The treatment can result in adecrease in seizure frequency and/or seizure severity, or a decrease inhigh gamma power activity by 10%, 20%, 30%, 40%, 50%, 60%, or 70%. Insome cases, the treatment can result in an improvement in cognitivefunction. Learning and/or memory can be improved by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100% or more than 100%.

Methods and compositions of this disclosure can be used to treat asubject who is at risk of developing a disease. The subject can be knownto be predisposed to a disease, for example, a neurological disease or adisease associated with epilepsy, seizures, and/or encephalopathy. Thesubject can be predisposed to a disease due to a genetic event, or dueto known risk factors. For example, a subject can carry a mutation inSCN1A which is associated with Dravet syndrome. In some cases thesubject can be predisposed to a disease such as Alzheimer's disease dueto the age of the subject.

The treatment can result in a decrease or cessation of symptoms. Forexample, treatment can improve learning, memory, cognitive function,and/or motor function; reduce frequency and/or duration of seizures;and/or reduce temperature sensitivity (or increase the temperaturethreshold for triggering a seizure).

In some instances, the target cell type of a gene therapy or expressioncassette disclosed herein is a PV cell. In some cases, the non-targetcell subtypes are at least one, at least two, at least three, or atleast four of the non-PV GABAergic subtypes disclosed herein. In somecases, an expression cassette comprising a regulatory element disclosedherein is selective for PV cells over all non-PV CNS cells. In somecases, cell-type selectivity is measured according to a co-localizationassay disclosed herein. In some cases, cell-type selectivity is measuredusing a mouse that expresses Cre in the target cell type.

In some instances, the treatment does not result in an adverse reactionfor the subject. Treatment with a gene therapy containing a PV-selectiveregulatory element can cause fewer, or less severe, adverse reactions ina subject than treatment with a similar gene therapy containing the sametransgene linked to a non-selective regulatory element.

In various aspects, any expression cassette disclosed herein can beadapted for or used in a gene therapy (e.g., rAAV or rAAV9 gene therapy)to treat any one or more of Dravet syndrome, Alzheimer's disease,epilepsy, neurodegeneration, tauopathy, neuronal hypoexcitability and/orseizure. In some cases, a gene therapy comprises any one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or ten or more of SEQ ID NOs: 1-32,or a functional fragment or a combination thereof, or sequences havingat least 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto, operably linked to a transgene. In somecases, a gene therapy comprises an expression cassette of thisdisclosure. In some cases, a gene therapy comprises one or moreregulatory elements disclosed herein operably linked to any transgene(e.g., a reporter transgene or a therapeutic transgene) such that theregulatory elements drive selective expression or preferentialexpression in at least one target cell type at a level that isstatistically significantly higher than the expression driven by CAG orEF1α or a non-selective regulatory element when operably linked to thesame transgene, or by the same construct without the regulatoryelements. In some cases, statistically significantly higher means theregulatory elements drive selective expression in the target cell typeat a level that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 times the expression level by the CAG, EF1α, aconstitutive promoter, or a non-selective regulatory element whenoperably linked to the same transgene in the target cell type, or by thesame construct without the regulatory elements. In some cases, suchcell-type selective expression is assayed using a co-localization assayas described herein. In some cases, the transgene is any one or more ofSCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, STXBP1, and afunctional fragment thereof. In some cases, the transgene is a DNAbinding protein that modulates expression of an endogenous gene, such asa transcriptional modulator, a transcriptional activator, or atranscriptional repressor. In some cases, the transgene is a DNA bindingprotein that comprises a DNA binding domain of a DNA binding protein ora DNA cleaving protein (e.g., a nuclease, a restriction enzyme, arecombinase, etc.) wherein the DNA cleaving domain or nuclease domainhas been deactivated, e.g., a nuclease-deactivated Cas (dCas), adeactivated transcription activator-like effector nuclease, or anuclease-deactivated zinc finger protein. In some cases, the DNA bindingdomain is linked to a transcriptional modulating domain (e.g., atranscriptional activator or repressor domain). In some cases, thetransgene is a gene editing protein, such as a zinc finger nuclease or atranscription activator-like effector nuclease. In some cases, atransgene is a reporter gene or a detectable marker, such as eGFP,tdTomato, or RFP. In some cases, a transgene is a Cas protein, such asCas9.

In some cases, a gene therapy comprising an expression cassettedisclosed herein is used to treat a neurological condition or disease.In some cases, a gene therapy comprising an expression cassettedisclosed herein is used to treat a neurological condition or disease,wherein the expression cassette comprises any one or more, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, or ten or more of SEQ ID NOs: 1-32, or afunctional fragment or a combination thereof, or sequences having atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence identity thereto, operably linked to a transgene. In somecases, the transgene is any one or more of SCN1A, SNC2A, SNC8A, SCN1B,SCN2B, KV3.1, KV3.3, STXBP1, a DNA binding protein and a functionalfragment thereof. In some cases, a gene therapy comprising an expressioncassette disclosed herein is used to treat Dravet syndrome. In someaspects, a gene therapy comprising an expression cassette disclosedherein is used to treat Alzheimer's disease. In some cases, a genetherapy comprising an expression cassette disclosed herein is used totreat epilepsy and/or seizure symptoms associated with Dravet syndromeand/or Alzheimer's disease. In some cases, treating any one of Dravetsyndrome, Alzheimer's disease, epilepsy, neurodegeneration, tauopathy,neuronal hypoexcitability and/or seizures comprises delivering oradministering a gene therapy of this disclosure to a cell of a subjectin need thereof. In some cases, the subject in need thereof is at riskfor or has any one of Dravet syndrome, Alzheimer's disease, epilepsy,and/or seizures. In some cases, the subject is a child or a minor. Insome cases, a gene therapy comprising an expression cassette disclosedherein is used to treat an infant, a child, or a minor diagnosed with oris at risk of developing Dravet syndrome. In some cases, a gene therapycomprising an expression cassette disclosed herein is used to treat asubject comprising a mutation or a genetic defect in any one or more ofSCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, and STXBP1.

In some aspects, the present disclosure provides a method of treatingany one of Dravet syndrome, Alzheimer's disease, epilepsy,neurodegeneration, tauopathy, neuronal hypoexcitability and/or seizures,comprising administering a gene therapy into a cell of a subject,wherein the gene therapy comprises an expression cassette disclosedherein. In some cases, such expression cassette comprises any one ormore of SEQ ID NOs: 1-32, or a functional fragment or a combinationthereof, or sequences having at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% sequence identity thereto, operably linkedto a transgene, wherein the transgene is any one of SCN1A, SNC2A, SNC8A,SCN1B, SCN2B, KV3.1, KV3.3, STXBP1, and a functional fragment thereof.In some cases, the transgene is a subunit of a sodium ion channel or apotassium ion channel. In some cases, the transgene is a syntaxinbinding protein. In some cases, the transgene is a transcriptionalmodulator, e.g., a transcriptional activator or a transcriptionalrepressor. In some cases, the transgene is a transcriptional modulatorthat modulates the expression of an endogenous gene (e.g., SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, or STXBP1). In some cases, atransgene is a gene editing protein, such as a zinc finger nuclease or atranscription activator-like effector nuclease. In some cases, thetransgene is a Cas protein, such as Cas9.

In other aspects, the present disclosure provides a method for modifyingany gene therapy designed for treating Dravet syndrome, Alzheimer'sdisease, epilepsy, neurodegeneration, tauopathy, neuronalhypoexcitability and/or seizures by adding one or more regulatoryelements disclosed herein to improve the cell-type selectivity of thegene therapy. In some cases, the gene therapy is an rAAV gene therapy.

In some cases, treatment with an expression cassette disclosed hereinreduces seizure duration and/or frequency, e.g., seizures associatedwith Dravet syndrome, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%as compared to an untreated control or as compared to the level beforetreatment.

In some cases, treatment with an expression cassette disclosed hereinreduces high gamma power activity (e.g., high gamma power activityassociated with Alzheimer's disease) by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% as compared to an untreated control or as compared to thelevel before treatment.

In one aspect, the present disclosure provides a nucleic acid cassetteof this disclosure comprises one or more regulatory elements operablylinked to a transgene that result in selective expression in a targetcell type over one or more non-target cell types, e.g., selectiveexpression in PV neurons in the CNS over one or more non-PV CNS celltypes. In some cases, each of the regulatory elements comprises (i) asequence of SEQ ID NOs: 1-32, (ii) a functional fragment or acombination thereof, or (iii) a sequence with at least 80% sequenceidentity to (i) or (ii). In some cases, the percent sequence identitycan be measured using BLAST. In some cases, at least one of theregulatory elements is human derived. In some cases, at least one of theregulatory elements is derived from a non-human mammal. In some cases,the regulatory elements are non-naturally occurring. In some cases, theregulatory elements result in a selectivity of expression in PV cellsthat is greater than expression of the transgene operably linked to CAGor EF1α or a non-selective regulatory element as measured by aco-localization assay. In some cases, a transgene is any one or more ofSCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, and STXBP1, or afunctional fragment hereof, or sequences having at least 80%, at least85%, at least 90%, at least 95%, or at least 99% sequence identitythereto. In some cases, a transgene is a DNA binding protein thatmodulates expression of a gene (e.g., an endogenous gene such as SCN1A,SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, and STXBP1), such astranscriptional modulator, transcriptional activator, or transcriptionalrepressor. In some cases, a transgene is a gene editing protein, such asa zinc finger nuclease or a transcription activator-like effectornuclease. In some cases, a transgene is a reporter gene or a detectablemarker, such as eGFP, tdTomato, or RFP. In some cases, a transgene is aCas protein, such as Cas9. In some cases, the regulatory elements resultin selective expression in PV cells at a level that is at least 0.5fold, at least 0.6 fold, at least 0.7 fold, at least 0.8 fold, at least0.9 fold, at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, atleast 1.4 fold, at least 1.5 fold, at least 2 fold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, atleast 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, atleast 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, atleast 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, atleast 90 fold, at least 100 fold as compared to that of a CAG or EF1α ora non-selective regulatory element, as measured by the co-localizationassay. In some cases, a fold difference refers to the fold differencebetween the percentage of eGFP+, PV+ cells that result from one or moreregulatory elements and that of a non-selective regulatory element. Insome cases, the co-localization assay is an immunohistochemical assay,as described below in Example 5. In some instances, a co-localizationassay is performed using a commercially available anti-PV antibody. Insome cases, the transgene encodes an ion channel subunit, aneurotransmitter regulator, or a variant or a functional fragmentthereof. In some cases, the ion channel subunit is an alpha subunit or abeta subunit of a sodium ion channel or a subunit of a potassium ionchannel. In some cases, the transgene is any one of (i) SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, or a DNA binding protein; (ii) afunctional fragment thereof; or (iii) a sequence having at least 80%sequence identity to (i) or (ii). In some cases, the neurotransmitterregulator is (i) STXBP1, (ii) a functional fragment thereof, or (iii) asequence having at least 80% sequence identity to (i) or (ii). In somecases, the regulatory elements and the operably linked transgene arelocated on different chromosomes. In some cases, the regulatory elementscombined are less than 2.5 kb, less than 1.5 kb, less than 1 kb, or lessthan 500 bp in size. In some cases, the non-PV cells comprise any one ormore of non-PV CNS cell types, including but not limited to excitatoryneurons, dopaminergic neurons, astrocytes, microglia, or motor neurons.In some cases, the nucleic acid cassette is a linear construct. In somecases, the nucleic acid cassette is a vector. In some cases, the nucleicacid cassette is a plasmid. In some cases, the vector is a viral vector.In some cases, the viral vector is an adeno-associated virus (AAV)vector. In some cases, the AAV vector is AAV1, AAV8, AAV9, scAAV1,scAAV8, or scAAV9. In some cases, the viral vector is a lentiviralvector. In some cases, the regulatory elements contain less than 600 bpof contiguous sequence from within 10 kb of the transcription start siteof GAD2, GAD1, SYN1, NKX2.1, DLX1, DLX5/6, SST, PV, and/or VIP.

In various embodiments disclosed herein, a regulatory element is lessthan 2050 bp, 2000 bp, 1900 bp, 1800 bp, 1700 bp, 1600 bp, 1500 bp, 1400bp, 1300 bp, 1200 bp, 1100 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp,500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50bp, 40 bp, 30 bp, 20 bp, 10 bp, or 5 bp. In various embodimentsdisclosed herein, an expression cassette comprises a transgene that islarger than a typical transgene size in a conventional viral vector,e.g., AAV. In some aspects, an expression cassette of any embodimentdisclosed herein comprises a transgene that is at least 1 kb, 1.5 kb, 2kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7kb, 7.5 kb, or 8 kb. In some aspects, any embodiment disclosed hereincomprises an expression cassette (e.g., AAV) that comprises a transgenethat is more than 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, or 4 kb in size. In some aspects, any embodimentdisclosed herein can further comprise one or more heterologous nucleicacid sequence or element.

In one aspect, a method of treating a neurological disorder or conditionin a subject in need thereof comprises delivering a therapeuticallyeffective amount of a nucleic acid cassette described herein. In anotheraspect, a method of increasing selective expression of a transgene in PVneurons comprises contacting a cell with a nucleic acid cassettedescribed herein. In some cases, a method of any embodiment disclosedherein is used to treat a neurological condition or disease, e.g., apsychiatric disorder (e.g., schizophrenia, obsessive compulsivedisorder, addiction, depression, anxiety, psychosis); an autism spectrumdisorder (e.g., Fragile X syndrome, Rett syndrome); epilepsy (e.g.,Dravet syndrome, chronic traumatic encephalopathy, generalized epilepsywith febrile seizures plus (GEFS+), epileptic encephalopathy, temporallobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration(e.g., Alzheimer's disease, Parkinson's disease). In some cases, amethod of any embodiment disclosed herein can be used to treat Dravetsyndrome. A method of any embodiment disclosed herein can be used totreat Alzheimer's disease. In some cases, methods and/or compositions ofthis disclosure can be used to treat any neurological condition ordisease associated with seizure and/or epilepsy, and/or wherein PVneurons are implicated.

In one aspect, a neurological condition described herein is treated witha gene therapy, preferably one that results in preferential expressionin one tissue type or cell type over another, e.g., a PV neuron asdetermined via a co-localization assay. In some cases, the gene therapyis an AAV.

In one aspect, a method of targeting expression of any transgene to PVneurons in the CNS comprises operably linking one or more of PV neuronselective regulatory elements to a transgene. In some cases, theregulatory elements comprise one or more sequences of SEQ ID NOs: 1-32,or sequences with at least 80% sequence identity to SEQ ID NOs: 1-32, ora functional fragment thereof. In some cases, the regulatory elementsresult in selective expression in PV neurons at a level that is at least2 fold, at least 5 fold, or at least 7 fold, or at least 10 fold ascompared to CAG or EF1α or a non-selective regulatory element operablylinked to the transgene, as measured by a co-localization assay. In somecases, the transgene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3,STXBP1, a DNA binding protein, or a functional fragment thereof. In somecases, the regulatory elements and the transgene are in an AAV. In somecases, the AAV is AAV9.

In one aspect, a method of treating a neurological condition or disorderin a subject in need thereof comprises contacting a cell with a nucleicacid cassette comprising one or more regulatory elements operably linkedto a transgene that results in selective expression in PV neurons overone or more non-PV CNS cells. In some cases, the regulatory elementscomprise one or more of SEQ ID NOs: 1-32, or a functional fragment or acombination thereof, or sequences having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity thereto. Insome cases, the transgene is a voltage-gated ion channel subunit, or avariant or a functional fragment thereof. In some cases, the subunit isa beta subunit of a sodium ion channel. In some cases, the subunit is analpha subunit of a sodium ion channel. In some cases, the subunit is ofa potassium ion channel. In some cases, the transgene is any one of (i)SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, a DNA binding proteinor STXBP1; (ii) a functional fragment thereof; or (iii) a sequencehaving at least 80% sequence identity to (i) or (ii). In some cases, theneurological condition or disorder is associated with ahaploinsufficiency or a mutation in any of SCN1A, SCN1B, SCN2B, KV3.1,and KV3.3. In some cases, the neurological condition or disorder isDravet syndrome. In some cases, the neurological condition or disorderis Alzheimer's disease. In some cases, the neurological condition ordisease is a psychiatric disorder (e.g., schizophrenia, obsessivecompulsive disorder, addiction, depression, anxiety, psychosis); anautism spectrum disorder (e.g., Fragile X syndrome, Rett syndrome);epilepsy (e.g., chronic traumatic encephalopathy, generalized epilepsywith febrile seizures plus (GEFS+), epileptic encephalopathy, temporallobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration(e.g., Alzheimer's disease, Parkinson's disease). In some cases, theneurological condition or disease is any seizure and/or epilepsy relatedcondition or disease wherein PV neurons are implicated. In some cases,the nucleic acid cassette results in selective expression in PV neuronsat a level that is at least 2 fold, at least 5 fold, or at least 7 fold,or at least 10 fold as compared to CAG or EF1α or a non-selectiveregulatory element operably linked to the transgene, as measured by aco-localization assay. In some cases, the nucleic acid cassette is in anAAV. In some cases, the AAV is AAV9.

In one aspect, a method of treating Dravet syndrome comprises contactinga cell with an AAV comprising a transgene that is any one of (i) SCN1A,SCN1B, SCN2B, a DNA binding protein (ii) a functional fragment thereof,or (iii) a sequence having at least 80% sequence identity to (i) or(ii). In some cases, the AAV further comprises one or more PV neuronselective regulatory elements or any of the regulatory elementsdisclosed herein operably linked to the transgene. In some cases, eachof the regulatory elements independently comprises a sequence comprisingany one of SEQ ID NOs: 1-32, or any functional fragment or combinationthereof, or a sequence comprising at least 80% sequence identity to anyone of SEQ ID NOs: 1-32.

In another aspect, a method of treating Alzheimer's disease comprisescontacting a cell with an AAV comprising a transgene is any one of (i)SCN1A, SCN2B, KV3.1, KV3.3, and STXBP1, (ii) a functional fragmentthereof, and (iii) a sequence having at least 80% sequence identity to(i) or (ii). In some cases, the AAV further comprises one or more PVneuron selective regulatory elements operably linked to the transgene.In some cases, each of the regulatory elements independently comprises asequence comprising any one of SEQ ID NOs: 1-32, or any functionalfragment or combination thereof, or a sequence comprising at least 80%sequence identity to any one of SEQ ID NOs: 1-32.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 Identifying Putative PV-Selective Regulatory Elements

To identify and screen putative regulatory element that are selectivefor PV cells, one can harvest PV cells from a R26-CAG-LSL-Sunl-sfGFP-Mycknockin mouse using affinity purification, e.g., using anti-GFP oranti-Myc antibodies and protein G-coated magnetic beads. PV cells can beenriched by using anti-PV antibody coated beads or affinity purificationmatrix. Nuclei are then isolated from the PV cells. Nuclear RNA can bepurified from the nuclei and converted to cDNA, and amplified with theNugen Ovation RNA-seq System V2 (Nugen 7102), followed by sequencingusing the Illumina HiSeq 2500. Genomic DNA can be purified from nuclei,fragmented, and used to make methylC-seq libraries, which can besequenced using the Illumina HiSeq 2000. To generate an ATAC-seqlibrary, nuclei bound to beads are transposed using Tn5 transposase(Illumina FC-121-1030). After 9-12 cycles of PCR amplification,libraries are sequenced using an Illumina HiSeq 2500. To generate aChIP-seq library, nuclei of PV cells are digested to mononucleosomesusing micrococcal nuclease, followed by salt extraction of chromatin,and native ChIP and library construction, which can be sequenced on anIllumina HiSeq 2500. After sequencing these libraries, the sequences aremapped to identify correlations and patterns in hypo-methylation inCG-rich regions, histone modifications, transcriptional factor bindingsites, and patterns associated with highly expressed transcriptionalfactors in PV cells. Overlapping features and correlations from multipleassays and/or libraries described above provide convergent evidence foridentifying candidate sequences that are putative PV-selectiveregulatory elements. Putative PV-selective regulatory elements can befurther tested using a co-localization assay as described in Example 5below. Putative PV-selective regulatory elements can also be tested inB6 PV-Cre mouse (Jackson Laboratory), which is a B6 PV-Cre knock-inmouse that expresses Cre recombinase in parvalbumin-expressing, asdescribed in Example 2 below. After validating PV-selectivity of theregulatory elements, the regulatory elements can be operably linked to atransgene to target expression selectively to PV cells over at leastone, two, three, four, five, or more than five non-PV cells.

Example 2 Selectivity for PV Neurons in PV-Cre Mouse

Selectivity for PV neurons can be determined using fluorescent imaging.AAV9 vectors containing eGFP operably linked to (i) a control promoter(EF1a); or (ii) a PV-selective RE identified in Example 1 above; or(iii) a PV-selective RE selected SEQ ID NOs: 1-32; and AAV9 vectorscontaining a Cre dependent tdTomato are co-injected into a B6 PV-Cremouse (Jackson Labs). PV-Cre is a knock-in mouse that expresses Crerecombinase in parvalbumin-expressing neurons (such as interneurons inthe brain and proprioceptive afferent sensory neurons in the dorsal rootganglia), without disrupting endogenous Pvalb expression.

Mice are infused bilaterally with 1.5 μL of AAV9 vector (5¹² to 1¹³gc/ml) into the dorsal and ventral hippocampus at a rate of 0.3 μL/minwith a 4 min rest period following injection. Mice are anesthetized forthe injection. The animals are placed in a stereotaxic frame (Kopfinstruments, USA), using the following coordinates for the dorsalhippocampus (AP −2.0 mm, lateral ±1.5, DV −1.4 mm from dura) and theventral hippocampus (AP −3.1 mm, lateral ±2.8, DV −3.8 mm from dura). AHamilton syringe (model #80308; 10 μL syringe with corresponding 30 gablunt tip needle) can be used with the stereotactic micromanipulator, todesignate and drill the bur holes. The drill is only used to penetratethe bone. Following drilling, the infusion cannula is lowered into thebrain to the depth of the desired location for injection, e.g.,injection volume: 1.5 μL; injection rate: 0.3 μL/min. Prior to infusion,the needle is allowed to equilibrate for 1 minute. Once delivery iscompleted, the needle is left for 4 min and then withdrawn overapproximately 1 min. Once all infusions are complete, the skin incisionis closed with sutures and administered post-surgery analgesics. Thetreated mice undergo daily health checks for the remainder of the studyand are weighed once weekly to monitor body weight.

For tissue collection, mice are euthanized via isoflurane overdose andperfused with 4% Paraformaldehyde (PFA). A piece of brain tissuecontaining the hippocampus is extracted and placed in 4% PFA at 4° C.for at least 12 hours. The brain tissue is then dehydrated in 30%sucrose (in phosphate buffered saline) at 4° C. until the tissue sinksto the bottom of the tube. Brain tissue is embedded in Tissue-Tek OCTfor sectioning in a cryostat. Sectioned brain tissue is stained for eGFPand tdTomato using standard immunohistochemistry procedures withanti-RFP polyclonal rabbit antibody (Rockland Antibodies and Assay) andanti-eGFP polyclonal chicken antibody (Ayes Labs). Fluorescencemicroscope imaging is used to visualize the cells. eGFP, or greenfluorescence, corresponds to all gene expression. Red fluorescence fromtdTomato corresponds to PV+ cells. An overlap of the two fluorescencesignals, which can be visualized as yellow or white cells, representsPV+ cells that express the eGFP transgene. AAV9 vectors comprising aPV-selective regulatory element is expected to yield higher number ofcells that are eGFP+ and PV+ as compared to the control promoter (EF1a).For example, fluorescence imaging of cells from mice injected with AAV9scomprising any one of PV-selective REs (e.g., SEQ ID NOs: 1-32 orputative REs identified in Example 1) are expected to show higher numberof eGFP+ cells that are also PV+. Selectivity for PV cells can bequantified as percentage of all eGFP+ cells that are also PV+.

Example 3 Reduction of Seizures in Dravet Mouse Model

B6(Cg)-Scn1a^(tm1.1Dsf)/J mice were obtained from the Dravet syndromeEuropean Federation via the Jackson Laboratories. These mice contain aDravet syndrome associated mutation in exon 24 of SCN1A (A to V atposition 1783). The mice also contain a floxed exon 24 with wildtypesequence. When not manipulated, this strain of mice expresses two copiesof the WT allele of SCN1A. However, upon delivery of an AAV expressingCre recombinase, any cell targeted by the AAV will switch to expressingone copy of the mutant allele. Upon expression of the mutant SCN1Asubunit, mice develop spontaneous seizures within 10 days.

B6(Cg)-Scn1a^(tm1.1Dsf)/J and control C57Bl6 mice were injected, as inExample 2, with AAVs expressing CRE recombinase under the control of theEF1α promoter and an AAV comprising PV cell selective regulatory elementSEQ ID NO: 32 driving expression of either eGFP (SEQ ID NO: 36) or SCN1B(SEQ ID NO: 37). Once all four infusions were complete, telemetryimplantation was performed immediately, (F20-EET, Data SciencesInternational). Electrocorticogram data was monitored continuously for14 days from 10 days after the surgery. Electrocorticogram data wasanalyzed and all seizure events were recorded, annotated with date, timestart, time stop, duration of the seizures, and severity score. FIG. 1illustrates the frequency of seizures in 12 hour windows over 14 daysfollowing treatment. The mice treated with SCN1B showed a trend towardslower seizure frequency compared to the control animals.

This observation was consistent with the notion that the beta unit ofthe sodium ion channel, e.g, SCN1B, can contribute to the traffickingand assembly of the sodium ion channel and that increasing theexpression of the beta unit selectively in PV neurons can result inincreased trafficking and assembly of the Nav1.1 channel, thus leadingto a trend towards lower seizure frequency and duration in the micetreated with SCN1B gene therapy.

Example 4 Treating Alzheimer's Disease in a Mouse Model

Female APP/PS1 and WT mice bred at PsychoGenics were used in the study.APP/PS1 mice contain human transgenes for both Amyloid Beta PrecursorProtein (APP) bearing the Swedish mutation (670 G-T and 671 A-C) andPresenilin 1 (PSEN1) containing an L166P mutation, both under thecontrol of the Thy1 promoter. These mice develop symptoms of Alzheimer'sdisease, including amyloid plaques and memory defects. Furtherdescription of these mice can be found in Radde et al, 2006 (Radde,Rebecca, et al. “Aβ42-driven cerebral amyloidosis in transgenic micereveals early and robust pathology.” EMBO reports 7.9 (2006): 940-946).

APP/PS1 mice were used as a model to determine the effect of treatmentwith SCN1B under the control of a RE on symptoms of Alzheimer's disease.APP/PS1 mice and non-transgenic controls were injected with either acontrol vector expressing eGFP or a treatment vector expressing SCN1B,both under the control of SEQ ID NO: 32; and implanted with an EETtransmitter as in Example 3. Brain activity was assessed over 24 hoursat 4 weeks after surgery. Electrocorticogram data was automaticallyanalyzed and power levels in the different frequency bands werecompared. FIG. 2 illustrates the high gamma power (50-100 Hz) innon-transgenic controls (WT), APP/PS1, and APP/PS1 mice treated withSCN1B. Increased high gamma power activity is associated with seizuresin Alzheimer's patients and epilepsy patients. The APP/PS1 mice showed ahigher level of high gamma power activity than the control mice.However, the increase was absent in the treated mice indicatingeffective treatment with the vector.

Example 5 Selectivity for PV Neurons in C57BL/6J (WT) Mouse

The selectivity of various REs disclosed herein were tested forselective gene expression in PV neurons using immunohistochemicalmethods. C57BL/6J (WT) mouse line was used for the PVimmunohistochemical assays. Expression cassettes comprising reportertransgene eGFP operably linked to a regulatory element (SEQ ID NO: 1 orSEQ ID NO: 8) or a CAG promoter in an AAV9 construct.

Pup Systemic Infusions:

Postnatal day 1 C57BL/6J mice were infused via facial vein injectionwith AA9 vector (1 E¹² to 3 E¹²) using a 300 U insulin syringe with a 31G needle. For tissue collection, mice were euthanized 21 dayspost-infusion via overdose of sodium pentobarbital (i.p.) and perfusedwith heparinized (2.5 IU/ml) saline followed by perfusion with 4%formaldehyde. Brains were removed and subsequently immersion-fixed in 4%formaldehyde for 24-48 hours at 4 degrees Celsius. The brain was thenplaced into PBS containing 30% sucrose and allowed to sink at 4 degreesCelsius (˜2-3 days). Upon sinking the individual brain hemispheres werefrozen in Tissue-Tek OCT with the midline facing down. Frozen brainswere processed for sagittal sections on a cryostat and placedfree-floating into PBS. Sections were stained for eGFP and parvalbumin(PV) using standard immunohistochemistry procedures with chickenanti-GFP (Ayes Lab, GFP-1020) and mouse anti-PV (Sigma, P3088).

Adult Systemic Infusions:

4-week-old, C57BL/6 mice were infused via tail vein injection with 60 μLof AAV9 vector (4.9¹³ to 1¹⁴ gc/ml) expressing eGFP. For tissuecollection mice were euthanized 21 days post-infusion via isofluraneoverdose and whole brains were extracted, washed with PBS and placedinto separate 5 ml tubes containing ice cold 4% formaldehyde. Tissue wasfixed at 4 degrees Celsius overnight. The following day, the brain wasplaced into PBS containing 30% sucrose and allowed to sink at 4 degreesCelsius. Upon sinking the individual brain hemispheres were frozen inTissue-Tek OCT with the midline facing down. Frozen brains wereprocessed for sagittal sections on a cryostat and placed free-floatinginto PBS. Sections were stained for EGFP and parvalbumin (PV) usingstandard immunohistochemistry procedures with chicken anti-GFP (AyesLab, GFP-1020) and mouse anti-PV (Sigma, P3088).

Immunohistochemistry Protocol:

Immunohistochemistry was used to analyze the co-localization of eGFPsignal and PV signal using the anti-PV antibody, wherein overlay of thesignals exhibited as white or light gray spots in the top panel images(merge), wherein representative overlay was indicated by arrowheads.Overlay of the eGFP and PV fluorescence is indicative of expression inPV cells. Such experiments can be used to determine the selectiveexpression of expression in PV cells. To perform the immunohistochemicalexperiments, tissues obtained from each mouse were blocked with aBlocking Buffer Solution (comprising 3% BSA, 3% NGS, 0.3% Triton X-100,0.2% Tween-20 in 1×PBS) for 1 hour at room temperature. The tissues werethen incubated with primary antibodies in blocking buffer overnight at 4C, washed three times with 1 mL 1×PBS, each with 5 minutes interval.Then the tissues were incubated with secondary antibodies in blockingbuffer for 1 hour at room temperature, followed by washing three times,each time with 1 mL 1×PBS and with 5 minutes interval. The tissues wereincubated DAPI (1:1000) in PBS buffer for 5 minute and wash twice with 1mL 1×PBS. Tissues were mounted onto slides, imaged, and analyzed using afluorescence microscope. Images were taken using a Vectra 3 imagingsystem (Perkin Elmer) and quantified for co-labeling of eGFP and PVstaining using inform-Tissue finder, advanced image analysis software orhand scored. At least 80 GFP positive cells were counted in each panelbefore determining the percentage of co-localization.

FIGS. 3A-3C illustrates the results of the immunohistochemistryexperiments performed in pups after systemic AAV9 injections. FIGS.4A-4C illustrates results of similar immunohistochemistry experimentsperformed in adult mice following AAV9 injections.

FIG. 3A illustrates the overlay of the immunohistochemistry experimentsperformed in pups after systemic AAV9 injections. FIG. 3B illustratesthe quantification of the co-localization of the immunohistochemistryexperiments, wherein selectivity for PV cells was measured as percentageof GFP+ cells that were also PV+, as compared to eGFP expression underthe control of the CAG promoter.

FIG. 4A illustrates the overlay of the immunohistochemistry experimentsperformed in adult mice after systemic AAV9 injections. FIG. 4Billustrates the quantification of the co-localization of theimmunohistochemistry experiments, wherein selectivity for PV cells wasmeasured as percentage of GFP+ cells that were also PV+, as compared toeGFP expression under the control of the EF1α.

It is estimated that GABAergic neurons constitute about 20% of CNS,while PV cells constitute about 40% of GABAergic neurons, which meansthat PV cells make up approximately 8% of all neurons in the CNS. SeePelkey, K A et al., 2017; and Lee, S. et al., 2010. Thus, one wouldpredict that about 8% of the cells labeled by a non-selective regulatoryelement (e.g., CAG, EF1α, or a constitutive promoter) would be PVpositive, or within this range. Therefore, expression in PV cells above8% is indicative of increased selectivity in PV cells. Notably, AAV9injections comprising regulatory element SEQ ID NO: 8 resulted in about60% of cells as PV positive, which was 7.5 times higher than what wasexpected by the distribution of PV cells.

Similar immunohistochemistry experiments as described above wereperformed to determine the selective expression of additional regulatoryelements, SEQ ID NOs: 2-7 and 9-22 as compared to a non-selectiveregulatory element having a sequence of SEQ ID NO: 34, except AAVDJviral vector was used to deliver eGFP operably linked to a regulatoryelement into C57BL/6J (WT) mice. Such AAVDJ virus was injected directlyinto the CNS in the hippocampus of adult mice. At least 80 GFP positivecells were counted in each experiment before calculating the percentageof co-localization, or selectivity, as percentage of GFP positive cellsthat were also PV positive. FIGS. 5A-5F illustrate the fluorescenceimaging used for determining co-localization, or selectivity, measuredas percentage of eGFP positive cells that were also PV positive and incomparison to the signal of non-selective regulatory element SEQ ID NO:34. Cells that were positive for a marker appear as white/gray cells inthe images. Merge images illustrate the overlap between thecorresponding eGFP and anti-PV images. Cells that were positive for botheGFP and PV appear as white/light gray cells in the merge image. FIG. 6illustrates the quantification of the co-localization analysis, measuredas percentage of eGFP+ cells that were also PV+.

Example 6 Treatment of Dravet Syndrome in Different Mouse Lines

Treatment of Dravet syndrome and/or symptoms thereof using theexpression cassettes described herein can be tested in various mouselines, such as B6(Cg)-Scn1a^(tm1.1Dsf)/J as described above,Scn1a^(tm1Kea), and Scn1a-R1470X mouse lines. These mouse lines areestablished mouse models for Dravet syndrome. Scn1a^(tm1Kea) andScn1a-R1470X mouse lines do not require CRE recombinase.

The Scn1a^(tm1Kea) mouse (available from the Jackson Laboratory;described in Hawkins et al., Scientific Reports, vol. 7: 15327 (2017))comprises a deletion of the first coding exon of SCN1A. Mice homozygousfor the SCN1A knockout allele are characterized by tremors, ataxia,seizures, and die by postnatal day 16. Heterozygous mice on the C57BL/6background develop spontaneous seizures and die within weeks. Such mousestrain can be used to study safety and efficacy of treatment of epilepsyand Dravet syndrome. See Miller et al., Genes Brain Behav. 2014February; 13(2):163-72 for additional information.

The Scn1a-R1470X mouse is a knock-in mouse carrying a premature stopcodon, R1407X, in exon 21 of the SCN1A gene. The same mutation has beenidentified as a pathogenic mutation in three unrelated SMEI patients.Scn1a^(RX/RX) pups are characterized by recurrent spontaneous seizuresat 12 postnatal days, including tonic-clonic and clonic seizures at12-16 postnatal days, and rhythmic jerking movements and involuntarymuscle contraction. See Ogiwara et al., Journal of Neuroscience, May 30,2007, 27 (22) 5903-5914 for additional information.

To test the compositions described herein, such as AAV gene therapy andtreatment using such gene therapy, Dravet mice of each of the mousestrains described above and control mice (e.g., a wild-type mouse or anuntreated Dravet mouse for the strain) are injected (e.g., administeredby intraperitoneal injection) with AAVs expressing either eGFP oranother reporter gene, or an expression cassette comprising one or morePV-selective REs SEQ ID NOs: 1-32) as described herein operably linkedto a transgene disclosed herein, such as SCN1A, SCN1B, or SCN2B, or anyof SEQ ID NOs: 37-39, or a variant or functional fragment thereof.Following AAV injections, mouse survival is monitored over time. Allmice are monitored daily for general health (e.g. weight, hydration,grooming, and mobility) and deaths were recorded. Telemetry implantationcan be performed immediately after AAV injections (F20-EET, DataSciences International). Electrocorticogram data can be recorded andmonitored continuously for at least 14 days from 10 days after thesurgery. All seizure events can be recorded for at least 14 daysfollowing AAV treatment, annotated with date, time start, time stop,duration, and severity score. A reduction in the frequency and/orduration of seizures following treatment with an AAV as described aboveas compared to the eGFP control or an untreated control is indicative ofthe efficacy of the gene therapy in reducing the symptoms and/orseverity of Dravet syndrome.

After treatment of the mice with AAV, the expression levels of thetransgene (e.g., SCN1A; SCN1B; SCN2B; a DNA binding protein, such as atranscriptional activator, that modulates an endogenous SCN1A, SCN1B, orSCN2B; any of SEQ ID NOs: 37-39; or any variant or functional fragmentthereof) can be monitored over time using various PCR and/or sequencingmethods to show AAV treatment can result in an increase in geneexpression in PV cells. Northern blot analysis and in situ hybridizationcan also be used to analyze transgene expression in vivo. The level ofthe protein expressed from the protein can also be monitored aftertreatment to show an increase in transgene expression correlates with anincrease in the corresponding protein in vivo. Protein levels can beassayed using various methods, including, but not limited to, Westernblot analysis, immunohistochemistry, immunofluorescence histochemistry,and/or ELISA assays. Formation of functional voltage-gated sodium ionchannels can also be assayed using current-clamp analysis.

Hyperthermia-induced seizures can be evaluated to compare the wild-typemice and/or untreated Dravet mice with Dravet mice treated with AAV genetherapy comprising an expression cassette described herein (e.g., anexpression cassette comprising one or more REs of this disclosureoperably linked to a transgene of this disclosure, such as SCN1A, SNC2A,SNC8A, SCN1B, SCN2B, a functional fragment thereof, or a DNA bindingprotein that modulates an endogenous SCN1A, SNC2A, SNC8A, SCN1B, orSCN2B). In such experiments, the core body temperature is monitored witha RET-3 rectal temperature probe (Physitemp Instruments, Inc, NewJersey, USA) and controlled by a heat lamp connected to a rodenttemperature regulator (TCAT-2DF, Physitemp) reconfigured with a Partlow1160+ controller (West Control Solutions, Brighton, UK). Bodytemperature is raised 0.5° C. every two minutes until the onset of thefirst clonic convulsion. As compared to the untreated Dravet mice,Dravet mice treated with an AAV gene therapy are expected to have ahigher threshold temperature before the onset of first clonic convulsionand/or have a higher proportion of mice that remain seizure free at themaximum temperature tested.

Different doses of AAV comprising an expression cassette can also beadministered to mice to determine the safety and efficacy profile ofeach gene therapy treatment. These preclinical studies can also informthe optimal dose(s) of the gene therapy to use for treating Dravetsyndrome.

Example 7 Treatment of Alzheimer's Disease in Mouse

Female APP/PS1 and wild-type (WI) mice, which are bred at PsychoGenicsand are established mouse model of Alzheimer's disease, can be used tostudy the safety and efficacy of the compositions described herein intreating Alzheimer's disease, comprising one or more PV-selective REs.APP/PS1 mice is describe above in Example 4.

APP/PS1 mice and non-transgenic controls are injected with either acontrol AAV vector expressing eGFP or a treatment AAV vector comprisingone or more PV-selective REs disclosed herein, e.g., SEQ ID NOs: 1-32,operably linked to a transgene that is deficient or impaired inAlzheimer's disease, such as SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, STXBP1,a DNA binding protein that modulates an endogenous gene (e.g., SCN1A,SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, or STXBP1), or any one of SEQID NOs: 37-43, or a functional fragment thereof.

Following AAV injections, mouse survival is monitored over time. Allmice are monitored daily for general health (e.g. weight, hydration,grooming, and mobility) and deaths were recorded. After injections ofthe AAVs, mice are also implanted with an EET transmitter as describedin Example 3 above. Brain activity can be recorded and monitored over 24hours for at least 4 weeks after surgery. Electrocorticogram data can beautomatically analyzed, and power levels in the different frequencybands (50-100 Hz) can be compared across different groups: WT mice,untreated APP/PS1 mice, and AAV-treated APP/PS1 mice, each treated withan AAV gene therapy as described above. Increased high gamma poweractivity is associated with seizures in Alzheimer's patients andepilepsy patients. Thus, the untreated APP/PS1 mice are expected to showa higher level of high gamma power activity than the control mice, whilethis increase is expected to be absent or reduced in the treated mice,indicating an effective treatment with an AAV gene therapy.

After treatment of the mice with AAVs, the expression levels of thetransgene can be monitored over time using various PCR and/or sequencingmethods to show AAV treatment can result in an increase in endogenousexpression of the transgene. Northern blot analysis and in situhybridization can also be used to analyze gene expression in vivo. Thelevel of the protein expressed from the transgene can also be monitoredafter treatment to show an increase in gene expression correlates withan increase in protein levels. Protein level can be assayed usingvarious methods, including, but not limited to, Western blot analysis,immunohistochemistry, and/or ELBA assays. Formation of functionalvoltage-gated sodium or potassium ion channels can also be assayed usingcurrent-clamp analysis.

Different doses of AAV comprising an expression cassette can also beadministered to mice to determine the safety and efficacy profile ofeach gene therapy treatment. These preclinical studies can also informthe optimal dose(s) of the gene therapy to use for treating Alzheimer'sdisease.

What is claimed is:
 1. A nucleic acid cassette comprising a regulatoryelement comprising SEQ ID NO: 8 operably linked to a heterologoustransgene.
 2. The nucleic acid cassette of claim 1, wherein theheterologous transgene comprises a nucleic acid sequence encoding a DNAbinding protein.
 3. The nucleic acid cassette of claim 2, wherein theDNA binding protein is a transcriptional activator that modulates anendogenous SCN1A gene.
 4. The nucleic acid cassette of claim 1, whereinthe nucleic acid cassette is an adeno-associated virus (AAV) vector. 5.The nucleic acid cassette of claim 4, wherein the AAV vector is AAV9 orscAAV9.
 6. The nucleic acid cassette of claim 3, wherein the nucleicacid cassette is an adeno-associated virus (AAV) vector.
 7. The nucleicacid cassette of claim 6, wherein the AAV vector is AAV9 or scAAV9. 8.The nucleic acid cassette of claim 1, wherein the regulatory elementcomprises SEQ ID NO:
 32. 9. The nucleic acid cassette of claim 8,wherein the heterologous transgene comprises a nucleic acid sequenceencoding a DNA binding protein.
 10. The nucleic acid cassette of claim9, wherein the DNA binding protein is a transcriptional activator thatmodulates an endogenous SCN1A gene.
 11. The nucleic acid cassette ofclaim 8, wherein the nucleic acid cassette is an adeno-associated virus(AAV) vector.
 12. The nucleic acid cassette of claim 11, wherein the AAVvector is AAV9 or scAAV9.
 13. The nucleic acid cassette of claim 10,wherein the nucleic acid cassette is an adeno-associated virus (AAV)vector.
 14. The nucleic acid cassette of claim 13, wherein the AAVvector is AAV9.
 15. The nucleic acid cassette of claim 13, wherein theAAV vector is scAAV9.
 16. A nucleic acid cassette comprising aregulatory element comprising a sequence with at least 95% sequenceidentity to SEQ ID NO: 8 operably linked to a heterologous transgenethat results in selective expression in parvalbumin (PV) neurons in theCNS over one or more non-PV cells in the CNS.
 17. The nucleic acidcassette of claim 16, wherein the heterologous transgene comprises anucleic acid sequence encoding a DNA binding protein.
 18. The nucleicacid cassette of claim 17, wherein the DNA binding protein is atranscriptional modulator of an endogenous gene.
 19. The nucleic acidcassette of claim 17, wherein the DNA binding protein comprises a zincfinger.
 20. The nucleic acid cassette of claim 17, wherein the DNAbinding protein is linked to a transcriptional activator domain.
 21. Thenucleic acid cassette of claim 17, wherein the DNA binding protein is atranscriptional activator that modulates an endogenous SCN1A gene. 22.The nucleic acid cassette of claim 16, wherein the nucleic acid cassetteis an adeno-associated virus (AAV) vector.
 23. The nucleic acid cassetteof claim 22, wherein the AAV vector is AAV9 or scAAV9.
 24. The nucleicacid cassette of claim 16, wherein the regulatory element comprises asequence with at least 95% sequence identity to SEQ ID NO:
 32. 25. Thenucleic acid cassette of claim 24, wherein the heterologous transgenecomprises a nucleic acid sequence encoding a DNA binding protein. 26.The nucleic acid cassette of claim 25, wherein the DNA binding proteinis a transcriptional activator that modulates an endogenous SCN1A gene.27. The nucleic acid cassette of claim 24, wherein the nucleic acidcassette is an adeno-associated virus (AAV) vector.
 28. The nucleic acidcassette of claim 27, wherein the AAV vector is AAV9 or scAAV9.
 29. Thenucleic acid cassette of claim 26, wherein the nucleic acid cassette isan adeno-associated virus (AAV) vector.
 30. The nucleic acid cassette ofclaim 29, wherein the AAV vector is AAV9 or scAAV9.